BIOL 205 Exam 1
What are ways that new genes can be introduced into target organisms to create transgenic organisms?
1) Electric shock, which induces holes in the cells' membranes and makes the cells more permeable, and more likely to take up transgenes into the cell. 2) Use viral vectors to insert gene of interest into the genome of a cell directly. 3) A way to transform plants is biolistics (or bioballistics, using a gene gun). where researchers coat gold particles with DNA plasmids containing the gene of interest. The DNA is delivered into the cell when the gold particles are shot through the cells' walls by a gene gun.
Give an example of three different small molecules that bind to proteins in cells and describe how they regulate protein function
1) The protein, CAP, which is an activator for the Lac operon must bind to another protein, CAMP, which allosterically alters CAP so that it can bind to its binding site on the DNA 2) GTP-binding proteins have their conformation and function altered when they hydrolyze GTP, releasing a phosphate, creating GDP. When GTP is bound to the binding protein, the protein is active, but when GDP is bound to the protein, it is inactive 3) Protein kinases phosphorylate proteins, which because phosphates are negatively charged, induces structural conformational changes of the protein, which changes function
What are the steps of colocalization using a transgene?
1. Introduce 2 transgenes or antibodies into the nucleus of the cell. One is going to be promoter + gene 1 + GFP and the other will be promoter + marker gene that is abundant in a known place + RFP. 2. Then, view the cells under the fluorescent microscope. 3. If you see red and green specks separately, this means that they do not co-localize, but if you see yellow specks, this suggests co-localization Using a transgene, we can use live cells
Describe the steps of a Western blot
1. Lyse the cells with detergent, sonication, mechanical force, or pressure. 2. Centrifuge the sample and pull out the supernatant, which contains the proteins 3. Denature the proteins with heat and mercaptoethanol and add SDS 4. Load the polyacrylamide gel with a ladder and then the total protein samples with different cell lines in different lanes 5. Transfer the proteins from the gel to the nitrocellulose paper that is more durable 6. Add a blocking agent (milk) so that there are no nonspecific interactions 7. Detect. Add a primary antibody that recognizes the protein of interest and then a secondary antibody that recognizes the primary antibody that is conjugated to a fluorophore
What are the steps of colocalization using an antibody?
1. Permeablize the membrane so the antibodies can get in (this kills the cells) 2. Introduce 2 antibodies, one for a marker and one for another protein. The antibodies will go find their protein and light up 3. Then, view the cells under the fluorescent microscope. 4. If you see red and green specks separately, this means that they do not co-localize, but if you see yellow specks, this suggests co-localization
Compare and contrast protein import into the ER and nucleus. Why might ER mechanism not work for the nucleus and vice versa
1. Proteins are imported into the nucleus after they have been synthesized, folded, and if necessary, assembled into complexes. In the ER, unfolded chaise are translocated as they are being made. Ribosomes are made in the nucleus and function in the cytosol and enzyme complexes that catalyze RNA transcription and splicing are assembled in the cytosol, but work in the nucleus, so both these need to be transported through the nuclear pores intact 2. Nuclear pores are always open to small molecules, where translocation channels in the ER are normally closed 3. Nuclear localization signals are not cleaved off after protein import, but ER signals usually are.
4 things that the light, florescent, scanning electron, and transmission electron microscopes have in common
1. Source of energy that will interact with the specimen 2. A component that promotes an interaction between the sample and the energy 3. Magnification 4. Detector
Conformation
3D shape of a protein based on the location of the atoms in relation to one another
What is a protein domain?
A conserved part of a given protein sequence and tertiary structure that can evolve, function and exist independent of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded.
Detergents are amphipathic molecules that are structurally very similar to phospholipids. Explain how exactly one could recognize a detergent molecule in a sea of phospholipids.
A detergent molecule have only 1 hydrophobic tail
What is a negative control and why is it necessary for a western blot?
A lysate from a cell line or tissue sample known not to express the protein you are detecting. This is to check for non-specific binding and false positive results.
What exactly is a biological membrane and why is it important?
A membrane that separates the cell and protects its chemical components from the outside environments. It is a site where signals are received and sites of selective permeability where molecules can be imported and exported
How does the number of peptide bonds of the backbone and side chains of amino acids in a polypeptide chain influence the shape and stability of the protein?
A noncovalent bond is not very strong, therefore it takes many noncovalent bonds to hold a polypeptide chain together
Small, water-soluble molecules pass freely through nuclear pores but larger molecules must gain entry to move through the pore. Describe how a larger macromolecule enters the nucleus.
A nuclear pore is a large complex composed proteins. Many of the proteins that line the nuclear pore contain extensive, unstructured regions in which the polypeptide chains are very disordered and the disordered segments form a tangled mesh that fills the center of the channel and prevents the passage of large molecules, but allows small and water soluble molecules to pass freely between the nucleus and the cytosol. To get into the nucleus, larger molecules must display an appropriate sorting signal called a nuclear localization signal that is recognized and bound by cytosolic proteins called nuclear import receptors. The receptors direct the protein to a nuclear pore by interacting with a fibril that extends from the rim of the pore into the cytosol, and disrupts the matrix so the pore proteins are not as tightly knit. When the nuclear import receptor + prospective nuclear protein enter the nucleus, it encounters Ran + GTP. Ran-GTP binds to the receptor, causing change its conformation and release its nuclear protein, and the receptor + Ran-GTP are transporter to the cytosol, and in the cytosol, an accessory protein triggers Ran to hydrolyze GTP into GDP + P. Ran-GDP falls off the import receptor, which is then free to bind another protein to bring into the nucleus, and this release of energy provides the energy for the import receptor to get another protein and enter the nucleus. PROTEINS ARE BROUGHT INTO THE NUCLEUS FULLY FOLDED
What is a positive control and why is it necessary for a western blot?
A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.
What is a nuclear import receptor?
A protein in the cytosol binds the nuclear localization signal on a protein and facilitates transport into the nucleus. Requires Ran-GTP binding to exit the nucleus.
How does secondary protein structure relate to quaternary protein structure?
A protein's secondary protein structure includes the a-helices and b-sheets that form within certain segments of the polypeptide chain, and quaternary protein structure is if the protein is formed as a complex of more than 1 polypeptide chain
What type of amino acid sequence is the most likely to form a transmembrane region of a transmembrane protein?
A sequence with mostly hydrophobic amino acids that can be stably integrated into the bilayer.
A protein that inhibits certain proteolytic enzymes (proteases) is normally secreted into the bloodstream by liver cells. This inhibitor protein, antitrypsin, is absent from the bloodstream of patients who carry a mutation that results in a single amino acid change in the protein. Antitrypsin deficiency causes a variety of severe problems because of the uncontrolled activity of proteases. Surprisingly, when a mutant antitrypsin is synthesized in the laboratory, it is as active as the normal antitrypsin at inhibiting proteases. Why then, does the mutation cause the disease? Can you suggest some mutants that would display these properties and explain why it would work in the laboratory but not in the body and how would you distinguish between the different mutants?
A single amino acid change causes the protein to misfold slightly, so that, even though it is still active, it is prevented by chaperon proteins in the ER from exiting in the organelle, and accumulates and is degraded. It could also be a mutation in the ER signal sequence that prevents it from entering the ER, because secreted proteins all enter the ER first It could also have an ER retention signal One could distinguish between these by using fluorescently tagged antibodies against the protein or expressing the protein as a fusion with GFP t follow its transport
What would happen to proteins bound for the nucleus if there were insufficient energy to transport them?
A single, incomplete round of nuclear import would occur Because nuclear transport is fueled by GTP hydrolysis, under conditions of insufficient energy, GTP would be used up and no Ran-GTP would be available to unload the cargo from its nuclear import receptor upon arrival in the nucleus. Unable to release its cargo, the nuclear import receptor would be stuck at the nuclear pore and not return to the cytosol to continue to import
In ion-exchange chromatography, what can we use to release a molecule bound to the column?
A solvent that alters the pH of the column because depending on the pH, a protein can have a net negative, net positive, or neutral charge. If the protein was negatively charged, it would bind to a positively charged column, but if you decreased the pH of the solvent, the negatively charged protein would pick up an H+ and no longer be attracted to the positively charged column and be eluted.
Proteins made in the cytosol on free ribosomes have their own "addresses," or sorting signals that take them to their target organelles. What is a sorting signal?
A sorting signal is an amino acid sequence that directs the protein to the organelle in which it is required. Proteins that lack a sorting signal remain in the cytosol
Which organism would have the highest percentage of unsaturated phospholipids in its membrane? A) Antarctic fish B) desert snake C) human D) polar bear E) thermophilic bacterium
A) Antarctic fish (Not the polar bear since they can regulate their own internal body temperature)
Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You wonder what happens to the protein in the mutant myocytes. A) Propose a testable hypothesis that could explain why pump function is altered in your mutant. B) Design an experiment that would allow you to test your hypothesis.
A) I hypothesize that the pumps are made, but the base pair substitution prevents them from from incorporating into the cell membrane, and they stay in the cytosol and cannot pump the calcium back in. The base pair substitution might have changed one amino acid from a nonpolar to a polar amino acid, and if this amino acid was one that normally is in the membrane spanning part of the protein, it could not incorporate into the membrane stably. B) I will use co-localization and attach GFP to the gene for the mutant pump and the promoter for the mutant pump and also use a marker gene of a protein that I know is in the ER membrane and plasma membrane that is fused with RFP and I will insert those into the nucleus of the cell in development and after the cell is big, I will look at the cell under a fluorescent microscope to see if my hypothesis is true. If my hypothesis is true, I will see the green in a different location than the red, meaning that the calcium pump is not in the membrane. I will see different colors becasue the fluorescent proteins will fluoresce at different wavelengths and thus emit different colors
Dr. Steinwand mutated the signal sequence (altered one out of 5 amino acids) that directs her favorite protein to the nucleus and found that it localized to the cytoplasm instead. This indicates that the wildtype signal sequence is: A) Necessary B) Sufficient C) Both D) Neither
A) Necessary (X is the signal sequence, and Y is going to the nucleus or not the nucleus.)
A simple enzyme reaction can be written by E+S <--> ES <--> E+P. A) Write a corresponding equation describing the workings of a transporter that mediates the transport of a solute down its gradient B) Why would this be an inappropriate description of channel function?
A) T + S <--> TS <--> T*S* --> T* + S* T <--> T* B) This does not describe the channel since solute passing through channels do not bind to them and channels do not undergo a conformational change in the process of transporting like transport proteins do
A transmembrane protein has the following properties: it has two binding sites, one for solute X and one for solute Z. The protein can undergo a conformational change and switch between two states: either both binding sites are exposed exclusively on one side of the membrane or both biding sites are exposed exclusively on the other side of the membrane. The protein can switch between the two conformational states only if both binding sites are occupied or if both are empty, but cannot switch if only one binding site is occupied. A) What kind of protein do these properties define? B) Do you need to specify any additional properties in order for this protein to move solute X up its concentration gradient and move solute Z down its electrochemical gradient?
A) This is a transporter that is a symport B) No, the important feature that provides the coupling of the 2 solutes is that the protein cannot switch its conformation if only one of the 2 solutes is bound. Solute B is driving transport of A, and B is in excess on the side of the membrane from which transport initiates and therefore occupies the binding site most of the time. In this state, the transporter is prevented from switching conformation until A binds.
Which of the following when abundant in the cytosol moves out of the cell down its concentration gradient but against its electrochemical gradient? A. K+ B. Cl- C. Na+ D. glucose
A. K+
You want to engineer a yeast cell to manufacture and secrete a bacterial protein product. To do this properly, you need to make certain that (circle all that apply): A. Plenty of clathrin-coated pits/buds will form B. The yeast cell is capable of endocytosis C. A hydrophobic C-terminal stop transfer sequence must be present D. The appropriate signal sequence is present E. The protein is amphipathic
A. Plenty of clathrin-coated pits/buds will form D. The appropriate signal sequence is present (You need clathrin to make sure that buds will form to pinch off and you need a signal sequence or else the protein will stay in the cytosol, and it needs to go to the ER to get placed into a vesicle through the endomembrane system because exocytosed proteins exit from the Golgi)
Which of the following hypotheses could be addressed by running a western blot on a fraction of cell homogenate/lysate isolated using differential centrifugation? Circle all that are correct. A. Protein A is targeted for degradation in the mutant and is thus less abundant in the mitochondria B. The size of protein A is altered in a mutant of interest C. Peroxisomes are more abundant in a mutant of interest
A. Protein A is targeted for degradation in the mutant and is thus less abundant in the mitochondria B. The size of protein A is altered in a mutant of interest (Western blots do not detect organelles)
Transporters undergo transitions between different conformations, depending on whether the substrate-binding pocket is empty or occupied. A. True B. False
A. True
Pumps are transporters that are able to harness energy provided by other components in the cells to drive the movement of solutes across membranes, against their concentration gradient. This type of transport is called: A. active transport B. facilitated diffusion C. passive transport D. free diffusion
A. active transport
Most animal fats form a solid at room temperature, while plant fats remain liquid at room temperature. Which of the following is a feature of lipids in plant membranes that best explains this difference? A. unsaturated hydrocarbons B. longer hydrocarbon tails C. higher levels of sterols D. larger head groups
A. unsaturated hydrocarbons
Phospholipid bilayers form sealed spherical vesicles in water. assume you have constructed lipid vesicles that contain Na+-K+ pumps as the sole membrane protein, and assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle. all the Na+-K+ pumps have the portion of the molecule that normally faces the cytosol oriented toward the outside of the vesicles. What would happen if you add ATP to a suspension in a solution containing both Na+ and K+ ions and had a solution with the same ionic composition inside them, but in addition to Na+-K+ pumps, the membrane of your vesicles also contains K+ leak channels
ATP becomes hydrolyzed and Na+ and K+ are pumped across the membrane, as normal. K+, however, immediately flows back into the vesicles through the K+ leak channels. K+ moves down the K+ concentration gradient formed by the action of the Na+-K+ pump. with each K+ that moves into the vesicle through the leak channel, a positve charge is moved across the membrane, building a membrane potential that is positve on the inside of the vesicles. eventually, K+ will stop flowing through the leak channels when the membrane potential balances the concentration gradient. the scenario described here is a slight oversimplification: the Na+-K+ pump in mammalian cells actually moves three sodium ions out of cells for each two potassium ions that it pumps into the cell, thereby driving an electric current across the membrane and making a small additional contribution to the resting membrane potential (which therefore corresponds only approximately to a state of equilibrium for K+ moving via K+ leak channels).
Phospholipid bilayers form sealed spherical vesicles in water. assume you have constructed lipid vesicles that contain Na+-K+ pumps as the sole membrane protein, and assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle. all the Na+-K+ pumps have the portion of the molecule that normally faces the cytosol oriented toward the outside of the vesicles. What would happen if half of the pump molecules embedded in the membrane of each vesicle were oriented the other way around so that the normally cytosolic portions of these molecules faced the inside of the vesicles. You then add ATP to the suspension
ATP would become hydrolyzed and Na+ and K+ would be pumped across the membrane as they normally would, however, the pump molecules that sit in the membrane in the reverse orientation would be completely inactive and they would not pump the ions the opposite way because ATP would not have access to the site on these molecules where phosphorylation occurs. this site is normally exposed to the cytosol. ATP is highly charged and cannot cross membranes without the help of specific transporters.
The neurotransmitter Acetylcholine is made in the cytosol and then transported into synaptic vesicles, where its concentration is more than 100x higher than in the cytosol. When synaptic vesicles are isolated from neurons, they can take up additional acetylcholine added to the solution, but only when ATP is present. Na+ ions are not requires for the uptake, but raising the pH of the solution in which the vesicles are suspended increase rate of uptake. Also, transport is inhibited when drugs are added that make the membrane permeable to H+ ions. Suggest a mechanism that explains this
Acetylcholine is being transported into the vesicles by an H+-Ach antiport in the membrane. The H+ gradient that drives the uptake is driven by an ATP-driven H+ pump in the vesicle membrane. Raising the pH of the solution outside decreases the H+ concentration, increasing outward gradient across the vesicle membrane
The budding of clathrin-coated vesicles from eukaryotic plasma membrane fragments can be observed with adaptins, clathrin, and dynamin-GTP are added to the membrane preparation. What would you observe if you omitted clathrin?
Adaptins still bind to the receptors in the membrane, but no clathrin coat can form, so no pits or vesicles are produced
Why can't bacterial cells be used to produce and accumulate genetically coupled immunotoxins?
Although the eukaryotic toxin does not inhibit its growth, they can only make immunotoxins that are not very complex, because they lack the ability to fold proteins with multiple domains or to form disulfide bonds between proteins
Differentiate between symporters and antiporters.
Although they are both active transporters called coupled pumps, but symports are coupled pumps that moves both solutes in the same direction across the membrane, and antiporters move solutes in opposite directions
Why was the algae C. reinhardtii able to be used to make immunotoxins?
Although they are eukaryotic, they have a chloroplast that takes up most of the cell and contain ribosomes and translational factors that resemble those of prokaryotes, but they contain a wide range of chaperone proteins, protein disulfide isomerases and peptidylprolyl isomerases that allow them to fold complex proteins
Proteins are built of what?
Amino acids
How does Na+ move into and out of cells?
An ATP driven sodium pump uses energy derived from ATP hydrolysis to transport sodium out of the cells and potassium into the cells because sodium constantly leaking in through other transporters and ion channels, and the pump keeps the sodium concentration in the cytosol 30x lower than in the ECF, creating a steep concentration gradient that tends to pull sodium back into the cell. Sodium also moves into the cells via coupled pumps
What is a coupled pump?
An active transport pump that links the uphill transport of one solute across a membrane to the downhill transport of another
What is the function of endosomes in cells?
An endosome is a membrane-enclosed compartment through which material ingested by endocytosis passes on its way to lysosomes. The endosomal compartment is a set of connected membrane tubes and larger vesicles that is kept acidic to cause receptors to release their bound cargo. It is the main sorting station on the inward endocytotic pathway.
Chloroplasts evolved from what?
An engulfed photosynthetic bacterium
Define feedback inhibition
An enzyme early in the reaction pathway is inhibited by a later product of that pathway so that whenever a large quantity of the product accumulates, the product binds to an earlier enzyme to shut down its activity
Why might a protein have two separate binding sites and how might the binding of a small molecule to one binding site influence the other binding site?
An enzyme might have more than one binding site, the active site that recognizes the substrates and other sites that recognize regulatory molecules. All of the sites communicate to cause the catalytic events at the active site to be controlled. The activity of the active site is changed by the binding at another site by conformational changes and the active site becomes less accommodating to the substrate molecule.
Explain how an mRNA molecule can remain attached to the ER membrane while individual ribosomes translating it are released to rejoin the cytosolic pool of ribosomes after each round of translation
An mRNA molecule is attached to the ER membrane by the ribosomes translating it. The ribosome population is not static, and the mRNA is continuously moved through ribosomes, and when a ribosome is done, it dissociates from the 3' end of the mRNA and the ER membrane, and rejoins the cytosolic ribosomes, but the mRNA remains bound to the other ribosomes that are still translating it
What kind of protein is used as a detector to probe the target protein in a western blot?
Antibodies
How would membrane fusion be different at 0˚ compared to 37˚?
At 0, the membrane fluidity would be reduced and the mixing would be slower
A single bacterial cell can live at various temperatures during its lifetime in part because it can alter the composition of its lipid bilayer. How would lipid composition differ when the bacterium finds itself in warmer temperatures vs when the bacterium finds itself in cooler temperatures?
At warmer temperatures, a cell makes membrane lipids with longer tails and with fewer double bonds so they are more viscous, but in colder temperatures, the membrane lipids will have shorter tails and more double bonds so they are less viscous.
CFTR is a transmembrane protein that moves Cl- ions down their concentration gradient without undergoing a conformational change. The movement of Cl- by CFTR is a form of: A) Simple diffusion B) Facilitated transport by a channel C) Facilitated transport by a transporter D) Active transport by a channel E) Active transport by a transporter
B) Facilitated transport by a channel (Channels do not change conformation, whereas transporters do)
What is the term used to describe the thick soup of cell extract produced when cells are broken open? A) Supernatant B) Homogenate C) Pellet D) Sample
B) Homogenate
Which of these amino acid sequences is most likely to form a transmembrane region of a transmembrane protein? A) I T L I Y F G N M S S V T Q T I L L I S B) L L L I F F G V M A L V I V V I L L I A C) L L K K F F R D M A A V H E T I L E E S
B) L L L I F F G V M A L V I V V I L L I A It contains primarily of hydrophobic amino acids and can be stably integrated into a lipid bilayer. In contrast, A contains many polar amino acids and C contains many charged amino acids which would be energetically disfavored in the bilayer
Which of the following is/are not a protein domain? A) ATP-binding site B) Light chain of an antibody C) Kinase D) Alpha helix E) Calcium binding site
B) Light chain of an antibody D) Alpha helix (The light chain of the antibody would not be able to bind an antigen without the heavy chain. The protein kinase domain is a structurally conserved protein domain containing the catalytic function of protein kinases. Protein domains are part of the tertiary structure and involve multiple alpha-helices or beta-sheets associating with one another. They can bee as simple as two alpha-helices wrapping around one another to form a coiled coil or be much more complex with multiple alpha-helices and beta-sheets involved. )
Which of the following amino acid sequences is most likely to encode a single pass transmembrane protein? A) Val-Leu-Leu-Asn-Lys-Ala-Met B) Ser-Asn-Tyr-Leu-Phe-Phe-His-Gln C) Asp-Val-Tyr-Met-Arg-Trp-Cys D) Gly-Ala-Val-Leu-Ile-Met-Trp-Phe
B) Ser-Asn-Tyr-Leu-Phe-Phe-His-Gln
Dr. Steinwand put the signal sequence that directs her favorite protein to the nucleus on a different protein that is normally localized to peroxisomes. When she did this, she found this other "other" protein in the nucleus of cells instead of peroxisomes! This indicates that the signal sequence is: A) Necessary B) Sufficient C) Both D) Neither
B) Sufficient (It isn't necessary, because if you were to take the signal sequence off of it, you do not know that it wouldn't normally go to the nucleus. We do not know what happens to Dr. Steinwand's protein once the signal sequence is taken off, so we do not know that it is necessary in order to direct the protein to the nucleus. If you have X, you get Y)
Your classmate ran a western blot, but had nothing appear in either lane. Which of the following is a plausible explanation for how they messed up? A) They forgot to add a blocking agent B) They forgot to add a primary antibody, and only used a secondary C) They forgot to add a secondary and only used a primary D) They used the pellet instead of the supernatant after their centrifugation E) They ran the wild type and mutant samples on the same lane F) They forgot to treat the samples with SDS and mercaptoethanol
B) They forgot to add a primary antibody, and only used a secondary C) They forgot to add a secondary and only used a primary D) They used the pellet instead of the supernatant after their centrifugation (Forgetting SDS would give them an unequal charge distribution, so the charge would affect how much they transfer, but we would still see bands)
Is Cl- or ethanol more likely to diffuse across the plasma membrane? A. Cl- B. Ethanol
B. Ethanol
Facilitated diffusion can be described as the favorable movement of one solute down its concentration gradient being coupled with the unfavorable movement of a second solute up its concentration gradient. A. True B. False
B. False
The net negative charge on the cytosolic side of the membrane enhances the rate of glucose import into the cell by a uniporter. A. True B. False
B. False (The net negative charge and low concentration of sodium on the cytosolic side enhances the rate of glucose import into the cell by a SYMPORTER)
Which of the following separates proteins according to size? A. Ion-exchange chromatography B. Gel-filtration chromatography C. Affinity chromatography
B. Gel-filtration chromatography
Your friend works in a biotechnology company and has discovered a drug that blocks the ability of Ran to exchange GDP for GTP. What is the most likely effect of this drug on nuclear transport? A. Nuclear transport receptors would be unable to bind cargo B. Nuclear transport receptors would be unable to enter the nucleus as GTP hydrolysis provides the energy for this process. C. Nuclear transport receptors would interact irreversibly with the nuclear pore fibrils
B. Nuclear transport receptors would be unable to enter the nucleus as GTP hydrolysis provides the energy for this process. (Would not be A, because once GTP is hydrolyzed to GDP, it dissociates from the nuclear import receptor)
Cheryl is performing SDS-PAGE gel electrophoresis but she forgets to heat her samples with SDS. Which of the following occurs as a result of Cheryl's mistake? A. Proteins separate by charge instead of by molecular mass B. Polypeptides do not unfold and thus migrate at slower rates through the gel C. Proteins do not migrate through the gel D. All of the above may occur as a result of Cheryl's mistake
B. Polypeptides do not unfold and thus migrate at slower rates through the gel
A bacterium is suddenly expelled from a warm human intestine into the cold world outside. Which of the following adjustments might the bacterium make to maintain the same level of membrane fluidity? A. Produce lipids with hydrocarbon tails that are longer and have fewer double bonds. B. Produce lipids with hydrocarbon tails that are shorter and have more double bonds. C. Decrease the amount of cholesterol in the membrane. D. Decrease the amount of glycolipids in the membrane.
B. Produce lipids with hydrocarbon tails that are shorter and have more double bonds. (bacteria do not have cholesterol in the membrane)
Molecules to be packaged into vesicles for transport are selected by: A. clathrin B. adaptins C. dynamin D. SNAREs
B. adaptins
We can test the relative permeability of a phospholipid bilayer by using a synthetic membrane that does not contain any protein components. Some uncharged, polar molecules are found to diffuse freely across these membranes, to varying degrees. Which of the following has the lowest rate of diffusion across an artificial membrane? A. water B. glucose C. ethanol D. glycerol
B. glucose (glucose is a very large, polar molecule)
Where does most new membrane synthesis take place in a eukaryotic cell? A. on ribosomes B. in the endoplasmic reticulum C. in the plasma membrane D. in the Golgi apparatus E. in the mitochondria
B. in the endoplasmic reticulum (On the surface of the ER by enzymes there. The ER synthesizes phospholipids and deposits the phospholipids in the cytosolic half of the bilayer, and an enzyme called a scramblase catalyzes the transfer of random phospholipids from one layer to another so the cell membranes grow evenly. Some of the newly assembled membrane will remain in the ER, but the rest will be used to supply fresh membrane to other organelles in the cell when bits of membrane get pinched off the form vesicles)
Proteins bind selectively to small-molecule targets called ligands. The selection of one ligand out of a mixture of possible ligands depends on the number of weak, noncovalent interactions in the protein's ligand-binding site. Where is the binding site typically located in the protein structure? A. on the surface of the protein B. inside a cavity on the protein surface C. buried in the interior of the protein D. forms on the surface of the protein in the presence of ligand
B. inside a cavity on the protein surface (Not going to be on the surface of the protein, because if it was on the surface, it wouldn't have as specific of a space to fit into)
The variations in the physical characteristics between different proteins are influenced by the overall amino acid compositions, but even more important is the unique amino acid ___. A. bond B. sequence C. orientation D. number
B. sequence
Some cells have aquaporins—channels that facilitate the flow of water molecules through the plasma membrane. For these cells, what regulates the rate and direction of water diffusion across the membrane? A. aquaporin conformation B. solute concentrations on either side of the membrane C. resting membrane potential D. availability of ATP
B. solute concentrations on either side of the membrane (channels move things by passive diffusion and osmosis is passive)
Proteins that are fully translated in the cytosol do NOT end up in: A. the cytosol B. transport vesicles C. the interior of the nucleus D. the mitochondria
B. transport vesicles (Ribosomes on the rough ER are actively synthesizing proteins that are delivered into the ER membrane or lumen)
An individual transport vesicle: A. contains only one type of protein in its lumen B. will fuse with only one type of membrane C. is endocytic if it is traveling toward the plasma membrane D. is enclosed by a membrane with the same lipid and protein composition as the membrane of the donor organelle
B. will fuse with only one type of membrane (Is not enclosed by a membrane with the same lipid and protein composition as the membrane of the donor organelle because think of the Clathrin-coated vesicles and Rab proteins)
Explain the role of phosphorylation in the regulation of protein function.
Because a phosphate group carries two negative charges, the addition of a phosphate group can cause a conformational change by attracting a cluster of positively charged side chains from another spot on the same protein, which will affect the binding of ligands elsewhere on the protein, thus altering the activity of the protein. Phosphorylation transfers a phosphate from ATP to an amino acid side chain of the target protein by a kinase
5 students in your class always sit together in the front row. This could either be because they really like each other or because nobody else in the class wants to sit next to them. Which explanation holds true for the assembly of the lipid bilayer? If the "wrong" explanation held true for the lipid bilayer, how would the properties of the bilayer be different?
Because nobody else in the class wants to sit next to them is the right answer because exclusion from water rather than attractive forces between lipid molecules is involved. If the lipids formed bonds with each other, the bilayer would be less fluid and fluidity is important becaue it enables membrane proteins to diffuse in the plane of the bilayer and interact with each other, it allows lipids and proteins to diffuse from sites where they are inserted after synthesis to other regions of the cell, it allows membrane molecules to be distributed evenly after the cell divides, and it allows membranes to fuse.
The sarcoplasmic reticulum stores calcium in muscle cells, keeping calcium levels low in the cytosol. When you flex your arm, calcium levels rise in the cytosol. To relax the cell after it contracts, calcium is removed from the cytosol by a pump in the SR membrane. Why do you think calcium pumps in the SR require ATP to function?
Because the SR stores calcium, the calcium concentration inside of the SR will be very high, so in order to move it back into the SR, we need energy to move it up its concentration gradient. In addition, the cell is negatively charged, so calcium will be drawn into the cell because of its electrical gradient as well
What happens to organelles as cells divide?
Before a eukaryotic cell divides, it must duplicate its membrane encased organelles. As cells grow, membrane enclosed organelles enlarge by incorporation of new molecules, the organelles then divide and during cell division are distributed between the two new daughter cells. Organelle growth requires a supply of new lipids to make more membrane and a supply of appropriate proteins, membrane protein and proteins that will occupy the interior of the organelle.
What is Bimolecular Fluorescence Complementation?
BiFC is a technique to see if protein A interacts with protein B. In order to do this, we will take protein A and fuse it to the N-terminus of GFP, and take protein B and fuse it to the C-terminus of GFP, then look under the fluorescent microscope and see if you see a signal. If you do see signal, this means that the proteins interact and that they have brought together the 2 halves of GFP, because the halves alone cannot fold properly, and thus cannot function, but when the halves are brought together, they will be able to fold properly and the fluorophore can be activated In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. This also allows us to see where the proteins interact as well.
What is a similarity and a difference between passive and active transport?
Both are mediated by membrane proteins, but passive transport is downhill and in the direction of its concenteation or electrochemical gradient. Active transport is uphill and needs an energy source. Active transport can be mediated by transporters, but not channels, whereas passive can be mediated by both
Explain how phosphorylation and the binding of a nucleotide (ATP or GTP) can both be used to regulate protein activity. What are the advantages of both forms of regulation?
Both nucleotide binding and phosphorylation can induce allosteric changes in proteins. An advantage of nucleotide binding is the fast rate at which a small nucleotide can diffuse to the protein, whereas phosphorylation is slower because the protein kinase needs to diffuse to the protein. Phosphorylation is advantageous because it only requires a ingle amino acid on the protein's surface rather than the specific binding site that nucleotides require, and phosphates can be added to many different side chains increasing the complexity of regulation
Predict the membrane orientation of a protein that is synthesized with an N-terminal cleaved signal sequence followed by a stop-transfer sequence, followed by a start transfer sequence.
Both the N and C terminus are in the ER lumen
Consider the descriptions below and select the one that can be classified as a protein domain: A) An alpha helix B) A complex of several polypeptide chain C) A substrate-binding pocket
C) A substrate-binding pocket
The signal recognition particle, SRP: A) Provides the energy for transferring a protein across the rER membrane B) Recognizes the 5' end of a mRNA coding for a secreted protein C) Inhibits translation by a free ribosome synthesizing a protein with a signal sequence D) Translocates proteins into the ER
C) Inhibits translation by a free ribosome synthesizing a protein with a signal sequence (There is no energy needed for transferring a protein across the rough ER membrane beasue they are being translocated as they are being made, so the elongation of the polypeptide provides the thrust needed. The SRP does not recognize the 5' end of the mRNA, it recognizes the N-terminus of the protein, and it does not translocate the proteins into the ER, that is the protein translocator)
Which order of magnitude corresponds to the size of a ribosome? A) Millimeters B) Micrometers C) Nanometers
C) Nanometers
Which of the following is used to purify proteins? A. Differential centrifugation B. SDS-page gel electrophoresis C. Chromatography D. Homogenization
C. Chromatography
Which of the following is NOT a characteristic of an antibody used in the process of western blotting? A. Specific to a protein of interest B. Attached to an enzyme that converts a color-less substrate to a colored substrate C. Migrates through the SDS-PAGE gel D. All of the above are characteristics of antibodies used in western blotting.
C. Migrates through the SDS-PAGE gel
Which of the following choices best describes the role of the lysosome? A. sorting of transport vesicles B. the storage of excess macromolecules C. clean-up, recycling, and disposal of macromolecules D. transport of material to the Golgi
C. clean-up, recycling, and disposal of macromolecules
Signal sequences that direct proteins to the correct compartment are: A. added to proteins through post-translational modification B. added to a protein by a protein translocator C. encoded in the amino acid sequence and SUFFICIENT for targeting a protein to its correct destination D. always removed once a protein is at the correct destination
C. encoded in the amino acid sequence and sufficient for targeting a protein to its correct destination (Signal sequences are added typically at the beginning of translation. They are also not always removed once a protein is at the correct destination, for example the start-transfer and stop-transfer signal sequences on transmembrane ER proteins are not removed and nuclear localization signals are not removed)
Which type of lipids are the most abundant in the plasma membrane? A. sterols B. glycolipids C. phospholipids D. triacylglycerides
C. phospholipids
The three-dimensional coordinates of atoms within a folded protein are determined experimentally. After researchers obtain a protein's structural details, they can use different techniques to highlight particular aspects of the structure. What visual model best displays a protein's secondary structures (α helices and β sheets)? A. wire B. backbone C. ribbon D. space-filling
C. ribbon
Which of the following phenomena will be observed if a cell's membrane is pierced? A. the membrane expands B. the membrane collapses C. the membrane reseals D. a tear is formed
C. the membrane reseals
Which of the following methods would be the most suitable to assess levels of expression of your target protein in different cell types? A. gel-filtration chromatography B. gel electrophoresis C. western blot analysis D. ion-exchange chromatography
C. western blot analysis (Gel electrophoresis would not necessarily work, since it would show all the proteins, not the target protein)
Rank the following in terms of diffusion rate: Methanol, K+, CO2, HCO3-, amino acids
CO2> methanol > amino acid > K+ = HCO3- (small non-polar > small polar > large polar > ions)
basic units of life that compose all living things.
Cells
List the three different approaches that the cell takes to regulate protein activity.
Cells can control how much protein it contains by regulating the expression of the gene that encodes the protein and regulating the rate at which the protein is degraded, cells control enzymatic activities by confining enzymes to particular subcellular compartments, proteins can be switched on or off
What is the technique that separates a homogenate into different parts?
Centrifugation
How can passive transport occur across a plasma membrane?
Channel, transport protein, or simple diffusion
Give a few examples of molecules that the membrane is highly impermeable to
Charged molecules because they are charged and have a strong electrical attraction to water. Hydrogen ions, sodium ions, potassium ions, calcium ions, chloride ions, magnesium ions, HCO3-
What process is used to purify proteins?
Chromatography
What technique would allow us to simply separate our favorite protein from the homogenate once cells are homogenized?
Chromatography
How do clathrin coated vesicles differ from COP-coated vesicles?
Clathrin coated vesicles bud from the Golgi on the outward secretory pathway and from the plasma membrane on the inward endocytic pathway. COP-coated vesicles are involved in transporting molecules from the ER to the Golgi, and from one part of the Golgi to another. In addition, the adaptins and other proteins that make the coated vesicles grab different receptor proteins and therefore different cargo, so they can help be selective
Pinocytosis mostly uses what type of vesicles?
Clathrin coated vesicles;
What is co-localization?
Co-localization answers the question of "where in the cell is my protein located?" It identifies the presence of two or more fluorescent labels in the same place in a cell, tissue, or organelle using a fluorescent microscope to see if two things are in the same place in the cell, but just because they are in the same place in the cell does not mean that they interact
What is the tertiary structure of a protein?
Covalent and non covalent interactions between the side chains to form the 3D structure
What type of bonds link together a polypeptide chain?
Covalent peptide bonds
How do proteins move from the polyacrylamide gel onto nitrocellulose?
Current is applied to flow the protein molecules from the gel to attach to the membrane; the positive electrodes are on the side of the membrane and the negative electrodes are on the side of the gel and the current will flow from negative to positive and as the current flows, the proteins flow to the nitrocellulose membrane
Mitochondria get their proteins from where?
Cytosol
What is the path that a protein travels from the cytosol to the lysosome?
Cytosol --> rough ER --> Golgi --> endosome --> lysosome
What is the path a protein travels from the cytosol to the plasma membrane?
Cytosol --> rough ER --> Golgi --> plasma membrane
Peroxisomes get their proteins from where?
Cytosol or from the ER
Margaret is a graduate student that studies root architecture in the model organism Arabidopsis thaliana. She has fixed and stained a mutant and a wild-type root with a non-fluorescent dye that adheres to the cell wall and would like to visualize the stained whole roots under the microscope. Which of the following microscopes should Margaret use to analyze her samples? A. A fluorescent microscope B. A scanning electron microscope C. An electron microscope D. A light microscope
D. A light microscope
Bob works in Margaret's lab but his studies focus on leaves. Bob would like to look at the surface details of a young leaf from Margaret's mutant. Which microscope would allow Bob to do this? A. An electron microscope B. A fluorescent microscope C. A light microscope D. A scanning electron microscope
D. A scanning electron microscope
Which of the following statements is TRUE? A. The polypeptide backbone is free to rotate about each peptide bond. B. Peptide bonds are the only covalent bonds that can link together two amino acids in proteins. C. The sequence of the atoms in the polypeptide backbone varies between different proteins D. Nonpolar amino acids tend to be found in the interior of proteins.
D. Nonpolar amino acids tend to be found in the interior of proteins. (Disulfide bonds are covalent bonds between -SH groups in the cysteine side chains)
__________ are fairly small organelles that provide a safe place within the cell to carry out certain biochemical reactions that generate harmful, highly reactive oxygen species. These chemicals are both generated and broken down in the same location. A. Endosomes B. Lysosomes C. Nucleosomes D. Peroxisomes
D. Peroxisomes
Which of the following is NOT TRUE of both mitochondria and chloroplasts and supports the theory of endosymbiosis? A. They contain their own DNA B. They reproduce by dividing in two C. They have their own ribosomes D. They manufacture energy-rich sugar molecules E. They were once bacterial cells that were engulfed by an early eukaryotic cell
D. They manufacture energy-rich sugar molecules
Although there are many distinct prokaryotic species, most have a small range of shapes, sizes, and growth rates. Which of the following characteristics are not observed in prokaryotes? A. the ability to divide rapidly B. a cell wall C. a highly structured cytoplasm D. endoplasmic reticulum
D. endoplasmic reticulum (a membrane enclosed organelle, and prokaryotes do not have membrane enclosed organelles)
Although all protein structures are unique, there are common structural building blocks that are referred to as regular secondary structures. Some proteins have α helices, some have β sheets, and still others have a combination of both. What makes it possible for proteins to have these common structural elements? A. specific amino acid sequences B. the hydrophobic-core interactions C. side-chain interactions D. hydrogen bonds along the protein backbone
D. hydrogen bonds along the protein backbone
Transporters, in contrast to channels, work by: A. filtering solutes by size B. filtering solutes by charge C. a gating mechanism D. specific binding to solutes
D. specific binding to solutes (they both have a gating mechanism)
What organelles are part of the endomembrane system?
ER, Golgi, peroxisomes, endosomes, lysosomes
The lipid bilayer of cell membranes is composed largely of phospholipids. Describe the composition of a single phospholipid molecule.
Each lipid molecule has a hydrophilic, phosphate-containing head and a pair of hydrocarbon hydrophobic tails. The phosphate is negatively charged, the choline is positively charged, and there is a glycerol molecule between the phosphate and the hydrophobic tails
How is vesicle origin and cargo identified within the cell?
Each type of transport vesicle in the cell displays molecular markers on its surface that identify the vesicle according to origin and cargo. Rab proteins are specific to each organelle and so each type of transport vesicle carries a unique combination of Rab proteins that serve as molecular markers for each membrane type.
Define membrane potential
Electrical imbalances that generates a voltage across the plasma membrane that is slightly negatie and this potential allows cells to power the transport of certain metabolites. There is not much of a charge difference, but there is a tiny one in the neighborhood of the plasma membrane
Explain the importance of fluidity in membrane function
Enables membrane proteins to diffuse rapidly in the plane of the bilayer and to interact with one another, permits membrane lipids and proteins to diffuse from the sites where they are inserted after their synthesis to other regions of the cell, ensures membrane molecules are distributed evenly between daughter cells when a cell divides, allows membranes to fuse with each other and mix their molecules
Describe the two different ways in which membrane-enclosed organelles arose
Endomembrane System- the nuclear membrane and the membranes of the ER, Golgi, endosomes, and lysosomes originated by invagination of the plasma membrane. The interiors of these organelles communicate extensively with one another and with the outside of the cell by small vesicles that bud off and fuse with each other. Endosymbiotic Theory-mitochondria and chloroplasts evolved from bacteria that were engulfed by the primitive eukaryotic cell with which they initially lived in symbiosis. Mitochondria and chloroplasts remain isolated from the vesicular traffic that connects the interiors of most of the other membrane-enclosed organelles to one another and to the outside of the cell
Mitochondria most likely evolved from what?
Engulfed aerobic bacterium
Promote intracellular chemical reactions by providing molecular surfaces with crevices that can cradle or exclude specific molecules; catalyze covalent bond breakage or formation
Enzymes
Defend the statement, "Enzymes act as catalysts."
Enzymes allow cells to make or break covalent bonds at will and enzymes are never used up in the reaction and always return to their normal form by the end
Why do eukaryotic cells require a nucleus as a separate compartment when prokaryotic cells can manage perfectly well without?
Eukaryotic gene expression is more complicated than prokaryotic gene expression, and prokaryotic cells do not have introns that interrupt the coding sequences of their genes, so that an mRNA can be translated immediately after it is transcribed. This would be dangerous in eukaryotic cells because most RNA transcripts have to be sliced before they can be translated and the nuclear envelope separates transcription and translation.
T/F: Animal cells have a cell wall
False
T/F: Lipid composition varies between the membranes of different organelles, but not among the leaflets of individual plasma membranes.
False
True or false? A large protein that will be localized to the nucleus that passes through a nuclear pore must have a Ran interacting domain.
False
T/F, The terms channel and transporter are used interchangeably in biology.
False (Channels are not as discriminatory as transporters, and only discriminate based on size and charge, while transporters bind very specific molecules and change conformation when they bind their substrate and channels are only in passive transport, transporters can be used in active or passive)
T/F: Saturated fats, like olive and peanut oil are typically liquids and room temperature
False (Unsaturated)
T/F, Membranes of different cell types contain the same membrane transport proteins.
False (each type of cell membrane has its own characteristic set of transport proteins, which determines exactly what solutes can pass into and out of the cell or organelle because different cells and organelles have different functions)
T/F: Facilitated diffusion is a form of transport that uses ATP to move solutes down their concentration gradients
False (only active uses ATP)
T/F, Transport protein A likely moves the same molecule as transport protein B across the membrane.
False (transport proteins are very specific and only transports proteins that fit into specific binding sites on the protein)
T/F: Antibodies can identify organelles
False!
True or false, a beta sheet can contain up to 5 strands, but no more
False, beta sheets can contain any number of strands because the two strands that form the rim of the sheet are available for hydrogen bonding to other strands. Remember that alpha helices and beta sheets are formed from hydrogen bonds in the backbone and that all polypeptide chains have the same backboe
True or false, affinity chromatography separates specific macromolecules according to charge
False, it separates them because of their interactions with ligands
T/F: N-linked sugar chains are found on glycoproteins that face the cell surface, as well as on glycoproteins that face the ER lumen, trans Golgi network, and mitochondria
False, mitochondria do not participate in vesicular transport and therefore N-linked glycoproteins that are assembled in the ER cannot be transported to the mitochondria
True/False: All proteins have quarternary structure
False, not all proteins form a complex of more than one polypeptide chain
T/F: Upon centrifugation, smaller organelles experience less friction and thereby sediment faster than larger ones
False, the larger an organelle is, the more centrifugal force it experiences and the faster it sediments, despite an increased frictional resistance from the fluid which it flows through
True or false, the specificity of an antibody molecule is contained exclusively in loops on the surface of the folded light-chain domain
False, the specificity is contained in the loops, but they are contributed by the light and heavy domains
T/F: The Na+/Glucose transporter moves glucose against its electrical and concentration gradient
False, there is no electrical gradient for glucose
T/F: Lysosomes digest only substances that have been taken up by cells via endocytosis
False, they digest internal organelles bt autophagy
T/F: Transporters allow solutes to cross a membrane faster than channels
False.
T/F: A symport would function as an antiport if its orientation in the molecule were reversed
False. A support binds two different solutes on the same side of the membrane. Turning it around would not change it to an antiport, which must bind different solutes on opposing sides of the membrane
T/F: The plasma membrane is highly impermeable to all charged molecules
False. The membrane contains transport proteins that confer selective permeability to many charged molecules. A pure lipid bilayer with no proteins is highly impermeable
True or false, the possible linear arrangements of amino acids are so vast that new proteins almost never evolve by alteration of old ones.
False. The possible linear arrangement of amino acids that lead to a stably folded protein domain are so few that most new proteins evolve by alteration of old ones
T/F: Channels have specific binding pockets for the solute molecules they pass
False. The selectivity of a channel is achieved by the size of the internal pore and by charged regions at the entrance of the pore that attract or repel ions
What is the purpose of a flow cytometry analysis?
Flow cytometry is a laser based technology employed in cell counting, cell sorting, biomarker detection by suspending cells in a liquid and passing them through a laser
Explain how the electrochemical gradient differs between Na+ and K+.
For sodium, its voltage and concentration gradients work together creating a very steep electrochemical gradient, but voltage and concentration gradients have opposing effects for potassium, and because of this, there is a small electrochemical gradient across the resting plasma membrane, so there is little net movement of potassium across the membrane even if the channels are open
Non-polar molecules move passively across the membrane but how do polar molecules move across the membrane? In other words, how do charge and concentration influence the direction of movement?
For uncharged molecules, the direction of passive transport is determined solely by its concentration gradient, but for electrically charged molecules, membrane potential affects the direction they move. The interior of the cell typically has a negative charge, so membrane potential tends to pull positively charged solutes into the cell and drive negatively charged ones out, but at the same time, a charged solute will also tend to move down its concentration gradient, and the net force driving a charged solute across a cell membrane is a composite of the 2 forces, one due to concentration gradient and the other due to the membrane potential, and the net driving force is called electrochemical gradient and it determines the direction that a solute will flow across the membrane by passive transport.
What are GTP binding proteins and how are they regulated?
GTP binding proteins are various types of proteins that can have GTP molecules bound to them and are active when GTP is bound, but can hydrolyze GTP to GDP and release a phosphate and flip the protein to its inactive form. GTP binding proteins can bind to other proteins and control their activities. They function as molecular switches in cells. The turning on and off of the switch is often stimulated in response to a signal received by the cell and GTP binding proteins in turn bind to other proteins to control their activities. The binding, hydrolyzing, and releasing of GTP all induces conformational changes on the G protein. Ran is an example of a GTP binding protein
Rab proteins
GTPases that are on the surface of each type of vesicle and recognized by corresponding tethering proteins on the cytosolic surface of the target membrane. Each organelle and each type of transport vesicle carries a unique combination of Rab proteins to serve as molecular markers for each membrane type
Enzymes tend to be what type of protein and why?
Globular because they fold into more compact shapes and do not tend to span long distances.
Sickle cell anemia is caused by a GluVal mutation in the hemoglobin gene. This mutation occurs at a site on the surface of the protein and promotes interactions between hemoglobin monomers that otherwise would not occur. As hemoglobin monomers interact, fibrils form and the protein's ability to carry oxygen is greatly reduced. What is the most likely explanation for how the GluVal mutation promotes fibril formation.
Glu is polar, and Val is nonpolar. A hydrophobic region is exposed when hemoglobin is mutated, and the hemoglobin start to come together and link together to hide the hydrophobic residue to tuck it on the inside so it is not exposed to the aqueous environment that the hemoglobin is in. The clustered hemoglobin proteins distort the red blood cells into a sickle shape
Invokana is a drug which inhibits the activity of sodium glucose symporters (SGLT) given to patients with diabetes. When Invokana is given, is glucose still able to enter cells at the same rate? What about sodium ions? Explain.
Glucose will not be able to enter the cell as quickly as it is a large molecule and the symporter greatly speeds up the rate of flow. Sodium is able to pass through ion channels and will still be able to flow into the cell as a similar rate.
Many proteins are glycosylated. What is glycosylation and what kind of function does it serve in the cell?
Glycosylation occurs in the ER, and it is the process of turning proteins into glycoproteins by the covalent attachment of a branched oligosaccharide chain. The oligosaccharides prevent the proteins from degradation, hold it in the ER until it is properly folded or help guide it to the appropriate organelle by serving as a transport signal for packaging the protein into transport vesicles. When they are on the cell surface, they are part of the glycocalyx and can function in the recognition of one cell by another. The first addition of the 14-sugar oligosaccharide in the ER is the first step in a series of modifations before the mature glycoprotein is made and it continues in the Golgi
Where do glycolipids acquire their sugar group?
Golgi
What does a scanning electron microscope allow us to see?
High resolution on the surface of something; like a 3D image
Is the backbone of a protein hydrophobic or hydrophilic?
Hydrophilic because they form alpha-helices and beta-sheet
Where in a folded protein in aqueous solution would one find polar amino acids? Non-polar side chains?
Hydrophobic (nonpolar) side chains will be clustered on the interior of the folded protein to avoid contact with the cytosol of the cell. Polar amino acids are found on the outside of the folded protein and they are hydrogen bonded to water and other polar molecules
Why is the formation of a lipid bilayer energetically favorable?
Hydrophobic molecules cannot readily dissolve in water because all of their atoms are uncharged and nonpolar, and cannot form favorable interactions with water molecules, and instead force adjacent water molecules to rearrange into an ordered cagelike structure around them, and because this cagelike structure is more highly ordered than the rest of the water, its formation requires free energy. The free energy is minimized when hydrophobic molecules cluster together because it limits their contact with the surrounding water molecules, which is why purely hydrophobic molecules tend to coalesce into a large drop when dispersed in water. The formation of the lipid bilayer is energetically favorable because the arrangement shields the hydrophobic tails from the water, while the hydrophilic heads are in contact with the water.
Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You hypothesize that the base pair substitution made it so the protein that needs to interact with the pump for the pump to work properly cannot interact. How would you test this?
I would use BiFC.
Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You hypothesize that the base pair substitution made it so the protein cannot fold properly, and it got degraded. How would you test this?
I would use Western blot comparing the wild type to the mutant, and use antibodies against the Ca2+ pump. If this hypothesis is true, I would expect little to no band in the mutant
Endosomes need an acidic lumen in order to function and that is achieved by an H+ pump in the endosomal membrane, which also contains Cl- channels. If channels do not function properly, acidification is also impaired. Why?
If H+ is pumped across the membrane, an electrochemical gradient of H+ results, which adds to the energy that is stored in the gradient and the energy that must be supplied to generate it, which limits transfer of more H+. If the membrane also has Cl- channels, the negatively charged Cl- that is concentrated in the cytosol will flow in and diminish the membrane potential, so it becomes less energetically expensive to pump more H+ in and the endosomes can be more acidic
Describe what you would expect to find in a well with low absorbance value after doing an MTT assay. Indicate relative population size of cells, color of medium, and other molecules present.
If a well had low absorbance, we would expect the opposite, since a low absorbance means that there was not a lot of colored salt, and if there is not a lot of colored salt this means that there was not much enzyme produced and if there was not much enzyme produced there was either not a lot of cells, or very inactive cells.
A rise in intracellular Ca2+ causes muscle cells to contract. In addition to an ATP-driven Ca2+ pump, muscle cells that contract quickly and regularly have an additional Ca2+ pump that is an anti port that exchanges Ca2+ for extracellular Na+. The majority of the Ca2+ ions that have entered the cell during contraction are rapidly pumped back out by this anti port. 2 heart drugs are used because they make the heart muscle cells interact more strongly and both of them function by inhibiting the Na+ pump. How do these work and what would happen if too much was taken?
If the Na+ pump was not working at full speed, the electrochemical gradient of Na+ that the pump generates is less steep, so the Ca2+-Na+ antiport will work less efficiently and Ca2+ is removed from the cell more slowly, so that when the next contraction begins, there is still some Ca2+ in the cytosol, so there is stronger contraction. Because the Na+ pump is essential, these drugs are deadly in high doses
How might a loss-of-function mutation in Ran affect the process of nuclear import?
If we lost the function of Ran, it would not bind to the nuclear import receptor, and it wouldn't cause the nuclear import receptor to release the protein by conformational changes to the import receptor since there are 2 binding sites (one for the protein and one for Ran) and the nucleus would not get proteins that it would need, and cells would die. The GTP hydrolysis by Ran drives the process forward by providing energy to get the receptor into the pore, and if Ran had a loss of function mutation, there would be no energy for nuclear import
Signal sequences are both necessary and sufficient. What does this mean and what are 2 experiments that justify this?
If we say that "x is a necessary condition for y," we mean that if we don't have x, then we won't have y. To say that x is a necessary condition for y does not mean that x guarantees y. If we say that "x is a sufficient condition for y," then we mean that if we have x, we know that y must follow. Necessary: if a signal sequence is deleted off of an ER protein, it converts it into a cytosolic protein. Sufficient if an ER signal sequence is placed at the beginning of a cytosolic protein, the protein is redirected to the ER
What is the purpose of using a marker gene in co-localization?
If you use a marker where you know where the protein found, it can help you identify certain places in the cell when it fluoresces because if not, it is hard to tell what is what within the cell
When a cell membrane has a small hole, it is able to quickly close in order to keep the hydrophobic tails away from the polar environment. Why might the membrane not be able to reseal in a detergent solution (think about the structure of a detergent molecule)?
In a detergent solution, the hydrophobic tails of the detergent will surround the hydrophobic tails of the membrane, making it impossible to form a complete membrane, and the phospholipids will not try to reassemble because the lipid-detergent micelles are stable in water because all of the hydrophobic sides are protected
Describe what you would expect to find in a well with high absorbance value after doing an MTT assay. Indicate relative population size of cells, color of medium, and other molecules present.
In a well with high absorbance values, we would expect a lot of cells that are very active, since they are making a lot of reducing enzymes that color the salt, and the more colored salt there is, the less the light passes through because it was absorbed. The medium would be highly colored with a lot of reducing enzyme in it
Explain why the polypeptide chain of most transmembrane proteins crosses the bilayer as an alpha helix or beta barrel
In both an alpha helix and beta barrel, the polar peptide bonds of the polypeptide backbone can be completely shielded from the hydrophobic environment of the bilayer by the hydrophobic side chains. Internal hydrogen bonds between the peptide bonds stabilize the helix and the barrel. Hydrogen bonding is maximized if they form a helix or barrel.
Where in the cell are phospholipids synthesized?
In eukaryotes, phospholipids are synthesized ON THE CYTOSOLIC SURFACR of the SMOOTH ER by enzymes which use free fatty acids as substrates and deposit the newly made phospholipids into the CYTOSOLIC HALF OF THE BILAYER
How would the output graph for flow cytometry differ for cells that have the target antigen binding to the primary and secondary antibody and for cells that DO NOT have the target antigen binding to the primary and secondary antibody?
In flow cytometry, before the antibodies are attached to the cells, a baseline run through is made, and a baseline peak is made for just the cells. Then, when the antibodies are added to the solution with the cells, the antibodies will bind to the cells that have binding sites for them, and when they are run through the laser, the peak shows up at a different spot because of fluorescent shift, since the fluorescent antibodies are now bound, and for the cells that do not have antibodies bound, their peak is at the same place as the baseline peak was.
Explain how the activation energy is lowered by a lysozyme during the hydrolysis of polysaccharides in the bacterial cell wall.
In order to sever the polysaccharide chains that form the cell walls of bacteria, the enzyme adds a molecule of water to a bond between 2 adjacent sugar groups in the polysaccharide, causing the chain to break, but without lysozyme, this reaction almost never occurs under normal conditions because in order for a water molecule to break a bond linking 2 sugars, the polysaccharide molecule has to be distorted to a particular shape, called the transition state, in which the atoms around the bond have an altered geometry and electron distribution, and to distort the polysaccharide in such a way requires a lot of energy from molecular collisions and normally, the energy of such collisions never exceeds the activation energy. Lysozyme holds the polysaccharide in its active site by forming many non covalent bonds, and lysozyme holds it in such a way that one of the sugars involved in the bond is distorted away from, its normal, stable conformation, which makes breaking the bonds more favorable, and when the breaking of the bonds is more favorable, there is a lower activation energy of the reaction and the polysaccharide in the bacterial cell wall gets severed. In addition, one of the amino acids in lysozyme polarizes the water molecule so that its oxygen can readily attach the polysaccharide
How did the v-SNARE get into the vesicle?
Internal signal sequence that got stuck in the ER membrane and around the same vicinity there was a cargo receptor
Predict the arrangement of a signal sequence that would enable the insertion of a multi-pass protein with an odd number of transmembrane segments
Internal start signal sequence, stop transfer sequence, and then another internal start transfer sequence. In this case, N will be in the cytosol, C will be in the lumen Cleaved N-terminal sequence, internal stop, start, stop, and in this case, N will be in the lumen, C will be in the cytosol
Why might it be advantageous to add a preassembled block of 14 sugar residues to a protein in the ER rather than building the sugar chains step bu stepson the surface of the protein by the sequential addition of sugars by individual enzymes?
It allows for better quality control. The assembled oligosaccharides can be checked for accuracy before they are added to the protein and if a mistake were made in adding sugars individually to the protein, the whole protein would be discarded
What is mercaptoethanol used in SDS-PAGE?
It breaks any -S-S- linkages between or within proteins so they will migrate at a rate that reflects their molecular weight
What is the path that a protein travels from the cytosol to the peroxisome?
It either goes straight from the cytosol, or for most proteins, to the ER and then to the peroxisome
We often think of cholesterol as a "bad" thing but it plays a very important role in the plasma membrane. What important role does cholesterol play in the plasma membrane?
It helps modulate the fluidity of the membrane because cholesterol molecules are short and rigid and fill the space between neighboring phospholipid molecules left by the kinks in their unsaturated hydrocarbon tails and therefore makes the membrane less fluid and less permeable
You have successfully identified a gene required for cell division in yeast called Cdc2. Yeast with loss-of-function mutations in Cdc2 cannot divide at warm temperatures. You are particularly interested in the protein encoded by Cdc2 because your data suggests that the causative mutation does not affect gene expression. In other words, you observed no difference in the relative abundance of RNA transcript in the mutant. What is a protocol that could test the hypothesis that the sub-cellular localization of the protein is altered in the mutant and thus it will be located in a different part of a mutant cell relative to that of a wild-type cell.
It is a protein involved in cell division, so the wildtype is in the nucleus To determine the sub-cellular localization of the altered protein, we will use a co-localization technique with antibodies. 1) We have 2 different plates of yeast cells with growth media. The first plate is the cells with the wildtype protein and the second plate has cells with the mutant protein GROWN IN AN INCUBATOR SINCE THE MUTANT IS TEMPERATURE SENSITIVE 2) First we will need to get antibodies that recognize and bind to the proteins (they will bind to both the wildtype and the mutant because the antibody recognizes such a small part of the protein that it probably will not differentiate between the wildtype and mutant that were made in a rabbit, and an anti-rabbit antibody that is tagged with a red fluorophore 3) Because the protein is involved in cell division and we know that the wildtype protein is in the nucleus, we will get an anti-transcription factor antibody conjugated to a green fluorophore. 4) We will permeablize the cells and allow the antibodies to enter and bind to their specific proteins 5) We will view the cells with a fluorescent microscope and the wildtype cell will serve as our control and we should see green and red colors together and in the mutant cells we should see the red in the nucleus, but green outside of the nucleus and this will tell us that the mutant is located in the incorrect part of the cell and that is why it is not working. To determine the sub-cellular localization of the altered protein, we will use a co-localization technique with a transgene 1) We have 2 different plates of yeast cells with growth media grown in an incubator.First plate is the cells with a transgene inserted into the nucleus that was promoter + WT Cdc2 + GFP. Second plate has cells with a transgene inserted into the nucleus that was promoter + mutated Cdc2 + GFP 2) Because the protein is involved in cell division and we know that the wildtype protein is in the nucleus, we will get an anti-transcription factor antibody conjugated to a red fluorophore. 3) We will view the cells with a fluorescent microscope and the wildtype cell will serve as our control and we should see green and red colors together and in the mutant cells we should see the red in the nucleus, but green outside of the nucleus and this will tell us that the mutant is located in the incorrect part of the cell and that is why it is not working.
What are immunotoxins?
It is a way to deliver drugs to specific cells; it consists of an antibody domain for binding target cells and molecules of a toxin that inhibit the proliferation of the targeted cell
What is the purpose of gel electrophoresis?
It is a way to sort and measure DNA strands and proteins
What is the Na+-K+ pump?
It is an active transporter found in the plasma membrane of animal cells that actively pumps Na+ out and brings K+ in using the energy derived from ATP hydrolysis
Describe what a tetrazolium salt assay is
It is colorimetric assay for assessing cell metabolic activity and can be used to measure cytotoxicity when exposed to a treatment molecule. In an MTT assay, growth medium and cells are plated, and then treatment molecules are added to the cells. Then, to see if the treatment molecule had any effect on the cells, tetrazolium salts are added to the plates. Tetrazolium salts are colorless when alone, but when the salt is exposed to electron acceptors, like those associated with reducing enzymes in cells, the salt reduces to form a colored dye, which limits the light that can pass through. Then, the plates are processed with a spectrophotometer that broadcasts light through the plate and an absorbance value is collected for the wells. The more dye produced, the higher the absorbance value and the number of active cells in each can be quantified according to the amount of dye produced.
Why do researchers usually use 2 antibodies?
It is more cost effective because we send the proteins to the rabbits to get the rabbits to make the primary antibodies, and they do not make fluorophores, and then if we have in the freezer, anti-rabbit antibodies that are not as specific, so they can be used more. They can also amplify the signal. If you release more secondary antibodies, more than 1 can bind to the primary antibody to amplify the signal
Dr. Outonalimb's claim to fame is her discovery of forgettin, a protein predominantly made by the pineal gland in human teenagers. The protein causes selective short-term unresponsiveness and memory loss when the auditory system receives statements like "Please take out the garbage!" Her hypothesis is that forgettin has a hydrophilic ER signal sequence at its C-terminus that is N-linked glycosylated in the ER and further glycosylated in the Golgi. She predicts that the protein is secreted from pineal cells into the bloodstream, from where it exerts its devastating systemic effects. You are a member of the committee deciding whether she should receive a grant for further work on her hypothesis. Critique her proposal.
It is questionable whether forgotten is a secreted protein because ER signal sequences are found at the N-terminus, and C-terminal hydrophobic sequences will be exposed outside the ribosome only after protein synthesis has terminated and cannot be recognized by an SRP during translation, so it is unlikely that it would be translocated by an SRP-dependent mechanism and will probably stay in the cytosol
What is SDS used for in a SDS-PAGE?
It is used to negatively charge the proteins because some amino acid side chains can be positively charged and it gives all of the proteins in the sample an even negative charge
How could the Golgi have evolved? For the Golgi to be functional, what else would be needed?
It may have evolved from specialized patches of the ER that pinched off to form a new compartment that still communicates with the ER by vesicular transport. Transport vesicles also are needed
What happens when a polar amino acid is buried within the protein?
It usually hydrogen bonds to the backbone or to other polar amino acids
When an early anaerobic pre-eukaryotic cell engulfed the aerobic bacterium that evolved into the mitochondria, how did the mitochondria/bacteria benefit?
It was protected, and it had access to the cytoplasm that was nutrient rich
Consider a protein that contains an ER signal sequence at its N-terminus and a nuclear localization sequence in its middle. What is the fate of this protein?
It will be translocated to the ER since its ER signal sequence is recognized as soon as it emerges from the ribosome, which then will become bound to the ER membrane and the growing polypeptide chain is transferred through the ER translocation channel. The nuclear localization sequence is never exposed to the cytosol and will never encounter nuclear import receptors
In a transmembrane protein destined for the ER, what arrangement of signal sequences would enable the insertion of a multipass protein with an odd number of transmembrane segments?
It would need a cleaved signal sequence, followed by an internal stop-transfer sequence, followed by pairs of start- stop-transfer sequences
The two halves of the lipid bilayer are called what?
Leaflets
What is the source of energy to interact with the specimen for the light microscope?
Light
Do prokaryotes have organelles?
Little to none
Glucose is stored in liver cells. Imagine the plasma membrane belongs to a liver cell and the human to which it belongs has just consumed a big donut. Glucose levels are high in the blood, and being transported into the cell for storage. Why is the sodium ion concentration gradient important for the transport of glucose into the liver cells?
Liver cells store glucose, so they have a lot of it in them, so it is not energetically favorable to move more glucose into the cell. We need the sodium gradient in order to bring the glucose into the cell. If we couple the transport of sodium with glucose, we can keep moving glucose from blood into cells
Thermus aquaticus is a bacteria that generally lives in very warm temperatures, often above 80°C. Describe what the phospholipids might look like in this organism's plasma membrane.
Long tails with few double bonds. This increases the amount of interactions between the tails and allows the membrane to maintain an appropriate amount of fluidity.
Give two examples of phagocytic cells and describe their role in our body. All eukaryotic cells are continually ingesting via pinocytosis, but large particles are ingested mainly by phagocytic cells.
Macrophages and neutrophils defend us against infection by ingesting invading microorganisms. To be taken up by macrophages or neutrophils, particles must bind to the phagocytic cell surface and activate a receptor, and some of these receptors recoegnize antibodies on bacteria, which will cuase them to engulf the bacteria and create a phagosome, which will then go and fuse with the lysosome. In addition, macrophages ingest dead and damaged cells and cell debris. These phagocytic cells extend sheetlike projections of their plasma membranes, called pseudopods to engulf the object via phagocytosis
The folded structure of a protein is stabilized by what?
Many non covalent interactions between different parts of the polypeptide chain
What method should you use to break open plant cells?
Mechanical force to shear the cells since the cell walls are very strong
The Na2+/Ca2+ antiport is responsible for removing Ca2+ from the cell by using the electrochemical gradient of Na2+. If Ca2+ is at a high enough concentration in the extracellular space, a symporter allows it to flow into the cell, bringing Mg+ in as well. When the Na2+/Ca2+ antiport is made inactive, will Mg+ be able to enter the cell at the same rate? Explain.
Mg+ will not be able to enter the cell in this situation. The Mg+ symporter relies on the antiporter to increase the Ca2+ gradient. If the Na2+/Ca2+ antiport is made inactive, calcium will not be removed from the cell and without the gradient bringing calcium in, the flow is no longer favored.
What is the role of a blocking agent in western blotting?
Milk is applied to cover most of the nitrocellulose membrane so the antibody doesn't bind to unspecific region of the membrane and because the antibody is so specific to the target, it will not let any blocking prevent it from binding. The nitrocellulose paper has a very high affinity for proteins (which makes sense since we could transfer proteins to it), and we want to block any unoccupied sites on the membrane
Define vesicular transport.
Movement of material between organelles in the eukaryotic cell via membrane-enclosed vesicles and the subsequent budding and fusion
List the following in order of increasing lipid bilayer permeability: RNA, Ca2+, glucose, ethanol, N2, H2O
N2 (small and polar), ethanol (small and slightly polar), water (Small and polar), glucose (large and polar), Ca2+ (charged), RNA (very large and charged...phosphate group)
The sodium-glucose pump is a common symporter. Which molecule is moving with its gradient and in what direction? What allows this pump to be energetically favorable?
Na+ is moving with its concentration gradient into the cell. The symporter uses the potential of the Na+ to allow glucose to flow into the cell, which is beneficial because cells have a low internal concentration of Na+ and are generally negatively charged, drawing the positive ions in.
Do all proteins have disulfide bonds?
No (Disulfide bonds are unstable in the cytosol)
Protein structure is determined solely by a protein's amino acid sequence. Would a genetically engineered protein in which the original order of all amino acids is reversed have the same structure as the original protein?
No becaue the peptide bond has a polarity. If you look at 2 amino acids in a polypeptide chain, the amino acid that is closer to the N-terminal end contributes the carboxyl group and the other amino acid contributes the amino group to to the peptide bond that links the two and changing the order would put the side chains in different positions with respect to the backbone and change the way the polypeptide folds (the shape of the folds involve atoms in the backbone and side chain)
Do co-localization see if two proteins interact?
No! Co-localization does NOT test protein-protein interactions. It addresses the question of where a certain protein is located in the cell
Can ions cross the plasma membrane freely?
No, charged
Once a protein has folded into its stable conformation, is its structure fixed?
No, it can change when the protein interacts with other molecules in the cell and the change in shape is crucial to the function of the protein
Can glucose cross the plasma membrane freely?
No, it is large and polar
If you want to study fragments of a protein, would you expect that any fragment of the polypeptide chain would fold the same way as it would in the intact protein?
No, many secondary structural elements are not stable in isolation, but are stabilized by other parts of the polypeptide chain. Hydrophobic regions of fragments which would normally be hidden in the inside of a folded protein would be exposed to water in an aqueous solution and in this case, they would aggregate nonspecifically and not have a defined structure and would be inactive for ligand binding.
Can ions diffuse through the plasma membrane? Why or why not?
No, since they are charged and polar, the hydrophobic tails are non polar and will repel charged molecules. The ion and its cloud of polarized water molecules can interact with the head, but not the tail
Can amino acids cross the plasma membrane freely?
No, they are large and often charged
Are proteins entering the ER folded?
No, they are unfolded because they enter the ER while being synthesized. Once they get in the ER, they are folded, associate with other proteins if necessary, get oligosaccharides attached, and form disulfide bonds if needed)
Are viruses considered living? Why or why not?
No, they need a host cell to replicate
Are adaptin proteins the same in every membrane?
No, this is one way that different organelles and membrane can select what type of molecules they want transported. The adaptins that bind cargo receptors in the plasma membrane are not the same as those that bind cargo receptors in the Golgi
Phospholipid bilayers form sealed spherical vesicles in water. assume you have constructed lipid vesicles that contain Na+-K+ pumps as the sole membrane protein, and assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle. all the Na+-K+ pumps have the portion of the molecule that normally faces the cytosol oriented toward the outside of the vesicles. Determine what would happen if your vesicles were suspended in a solution containing both Na+ and K+ ions and had a solution with the same ionic composition inside them?
Nothing, you require ATP to drive the pump
Do proteins unfold to enter the nucleus? The chloroplast? The peroxisome?
Nucleus- Unlike most other organelles, nuclear pores transport proteins in their fully folded conformation. Chloroplast & mitochondria- proteins are unfolded as they are transported, and once they are inside, chaperone proteins help them to fold. Peroxisome-proteins do not need to unfold
Why is horseradish peroxidase conjugated to secondary antibodies?
Once a substrate is added, the enzyme converts the substrate into a product and the product that appears is colored
How might an early, anaerobic eukaryote have benefited from taking up a bacterium (prokaryote)?
Once the anaerobic eukaryote took up the photosynthetic bacterium, the photosynthetic bacterium evolved into the chloroplast so that the anaerobic eukaryote can now use its energy to make food. Once it took up the aerobic bacterium, they can now use oxygen to produce, which is much more efficient for energy production
Knowing that TAS2R2 is a receptor for cyclohexamine, how could Dr. Dorian test to see if the ligand binding site of the mutated protein is dysfunctional?
One possible solution is to use affinity chromatography with beads conjugated to cyclohexamine. Because TAS2R2 would bind to the cyclohexamine, it would slowly precipitate out in relationship to other proteins and could be isolated* from the wild type. Then, by running another chromatograph with the mutant sample, he could see if the protein precipitates in the same time frame (if at all). If the protein does not separate from the rest of the sample, then the mutation likely affects the binding site. If it precipitates like the WT, then the mutation occurs somewhere else in the protein. Then, a western blot would be performed to verify that TAS2R2 was the protein that separated
Glycolipids can be found on which side of the phospholipid bilayer?
Only on the noncytosolic side
After purifying proteins through chromatography, how can you assess purity?
PAGE
Provide 3 pieces of evidence that support the endosymbiotic theory
Paramecium bursaria is a modern single-celled eukaryote that swallows green algae, but does not digest them. They swim to the light to let the algae photosynthesize and has an endosymbiotic relationship with the algae by providing the algae with carbon dioxide, nitrogen components, and protection and in return, the algae supplies their host with maltose Mitochondria were probably early aerobic prokaryotes and this is because they contain their own circular DNA chromosome and reproduce on their own by binary fission Chloroplast were probably early photosynthetic prokaryotes and this is because they contain their own circular DNA chromosome and reproduce on their own by binary fission
How are paramecium bursaria evidence for the endosymbiotic theory?
Paramecium bursaria, a single-celled eukaryote that swims around in pond water, may not have its own chloroplasts, but it does manage to "borrow" them in a rather unusual way. P. bursaria swallows photosynthetic green algae, but it stores them instead of digesting them. When P. bursaria swims into the light, the algae photosynthesize sugar, and both cells share food. It is evidence since it shows that the single cell engulfs another cell and holds on to it for a mutual, beneficial relationship
Microtubule
Part of the cytoskeleton that serves as spindle fibers in cell division to pull the chromosomes apart and made of the protein, tubulin
Why does a polypeptide cross the membrane in the form of an alpha-helix or beta-sheet?
Peptide bonds that join amino acids in the protein are normally polar, making the polypeptide backbone hydrophilic. Because water is absent from the interior of the of the bilayer, atoms forming the backbone are driven to form hydrogen bonds with one another and hydrogen-bonding is maximized if the polypeptide chain forms an alpha helix. In the membrane-spanning alpha helices, the hydrophobic side chains are exposed on the outside of the helix and the polypeptide backbone forms hydrogen bonds with one another on the inside of the helix. For the beta-sheet, the polypeptide is rolled into a cylinder where the amino side chains face the inside of the barrel if they are hydrophilic and the amino acid side chains that face the outside of the barrel are hydrophobic. When the protein folds into a helix or sheet, the polypeptide backbone is shielded from the hydrophobic lipid environemtn of the membrane by its protruding nonpolar side chains
Compare and contrast phagocytosis and pinocytosis.
Phagocytosis is cellular eating and the ingestion of large particles like microorganisms and cellular debris and pinocytosis is cellular drinking, which is the ingestion of fluid and small molecules.
What are the differences between a phospholipid molecule and a detergent molecule? How would the structure of a phospholipid need to change to make it a detergent?
Phospholipid molecules are cylindrical in shape and detergent molecules are conical shaped. A phospholipid molecule with one tail would be a detergent. To make a phospholipid a detergent, we either need to make the head bigger or remove a tail.
Define endocytosis.
Process by which eukaryotic cells take in materials through the invagination of the plasma membrane which h surrounds the ingested material in a membrane enclosed vesicle and specialized cells within the eukaryote can take in material this way
You are looking through a microscope at a cross section of a cell and notice irregularly shaped tubes, sacs, and spheres distributed throughout the cell. Why do these structures suggest to you that this particular cell is eukaryotic?
Prokaryotic cells do not have membrane enclosed organelles like eukaryotic cells do, and prokaryotic cells consist of a single compartment enclosed by a single plasma membrane
The budding of clathrin-coated vesicles from eukaryotic plasma membrane fragments can be observed with adaptins, clathrin, and dynamin-GTP are added to the membrane preparation. What would you observe if you used prokaryotic cell membrane fragments?
Prokaryotic cells do not undergo endocytosis, so they do not have the appropriate receptors
Peroxisome
Protective environment where toxic things are broken down because hydrogen peroxide is used to inactivate toxic molecules
What does it mean to purify proteins?
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture
How are proteins transported into the mitochondria and chloroplast?
Proteins destined for the mitochondria or chloroplast have a signal sequence on their N-terminus that allows them to enter. To enter, the protein signal sequence is recognized by an import receptor protein on the outer membrane that is associated with a protein translator, and this complex diffuses laterally in the outer membrane until it encounters a 2nd translocator in the inner membrane and the 2 translocators transport the protein across the two membranes, unfolding the proteins in the process. Once the protein is inside, the signal sequence is cleaved off by a signal peptidase and chaperone proteins help them fold into their final conformation
In ion-exchange chromatography, what binds to the column?
Proteins of the opposite charge of the beads
In gel-filtration chromatography, what binds to the column?
Proteins that can fit into the pores; smaller molecules
Antibodies are globular proteins composed of 4 polypeptide chains, two of which are held together by a disulfide bond. What level of organization would be used to describe the structure of an antibody and why?
Quarternary since it is composed of more than 1 polypeptide chain
An antibody is composed of four polypeptide chains that are held together by disulfide bonds. What level of protein structure does an antibody have?
Quaternary structure
How are quaternary structures held together?
Quaternary structures are held together by the same types of chemical bonds that are found in tertiary structure, including a variety of weak bonds and disulfide bridges.
What is the protein, Ran?
Ran is a GTP (nucleotide) binding protein. It hydrolyzes bound GTP in order to provide energy for nuclear import and binds to the nuclear import receptor causing it to change its conformation and release the protein into the nucleus, and helps the nuclear import protein exit the nucleus
Many proteins including those required for mitosis must be transported into the nucleus from the cytosol. Propose an explanation as to why scientists have proposed using inhibitors of Ran function to treat cancer.
Ran is necessary for nuclear import by hydrolyzing GTP to provide the energy needed for transport into the nucleus, and proteins required for mitosis are transported from the nucleus from the cytosol and in cancer, cells divide uncontrollably and if we can prevent some proteins required for mitosis from getting to the nucleus, the proteins required for mitosis will not be getting into the nucleus and so cells cannot divide to slow cancer progression. This could be bad because it might not just target cancer cells
Peroxisomes contain enzymes that function in the breakdown of toxins, alcohols, and fatty acids. Receptors involved in the import of proteins localized to peroxisomes are unique in that they are not anchored to the peroxisome's membrane. Where are the receptors for peroxisome localized proteins found in the cell?
Receptors proteins that recognize proteins destined for peroxisomes are found in the cytosol, which bring them to a protein translator in the peroxisomal membrane, but peroxisomal proteins do not need to be unfolded
Lysosome
Recycling center for the cell where things are broken down. It is the intracellular digestion center where nutrients are released from digested food particles or are excreted from the cell
Hydrogen bonds between neighboring regions of the polypeptide backbone give rise to what?
Regular folding patterns, known as alpha helices and beta sheets
Describe the role of SNAREs in the docking and in the fusion of a vesicle and in membrane coalesce.
SNAREs interact with the complementary SNARE to dock the vesicle into place. SNAREs also catalyze the membrane fusion required for a transport vesicle to deliver its cargo and add the vesicle membrane to the membrane of the organelle. After docking, fusion requires the two bilayers to come so close that their lipids intermix and water must be displaced from the hydrophilic surfaces of the membranes. The displacement of water is energitally unfavorable and prevents membranes from fusing randomly, so the fusion process must be catalyzed by SNARE proteins. After the fusion is catalyzed, t-SNAREs and v-SNAREs wrap around each other and act like a winch that pulls the two lipid bilayers close. ALL TRANSPORT VESICLES MUST HAVE A V-SNARE IN THEIR MEMBRANE
What does a transmission electron microscope allow us to see?
See the inside of the cell in fine detail
What give amino acids their unique properties?
Side chains (the backbones are all repeating sequence of N-C-C with peptide bonds between them)
Signal-recognition proteins (SRPs) present in the cytosol guide proteins with ER signal sequences bound to free ribosomes to the ER membrane. Describe how an SRP facilitates the transport of a soluble protein to the ER lumen.
Signal recognition particles bind to both the ribosome and the ER signal sequence and slows the protein synthesis, so that the SRP has time to engage with an SRP receptor within the membrane of the ER. Once the SRP is bound to the SRP receptor, the SRP is released and the receptor passes the ribosome to a protein translocator that is also in the ER membrane and protein synthesis continues and the polypeptide chain is threaded across the ER membrane through a channel in the protein translocator. The signal sequence functions to open the channel of the protein translocator, and this sequence remains bound to the membrane while the rest of the protein is being threaded through. It is removed by a transmembrane signal peptidase
How can the serine proteases be nearly identical in sequence and structure, yet have different functions?
Similar sequences will allow the proteins to fold into similar tertiary shapes, and make the fold and have the catalytic residues needed to be a proteases, but what parts on the protein they bind things and their properties can be affected by the small differences in amino acids between them. Because enzymes bind so tightly to their substrates, just a small change in shape can affect what substrates serine proteases can bind to
Name two properties of molecules that diffuse rapidly across biological membranes
Small and hydrophobic
For red blood cells, how does cell shape fit function?
Small, flexible, circular to move easier through the vessels in our body Greater surface area: volume ratio They do not have a nucleus, so this means that they have more space for hemoglobin to bind to more hemoglobin protein, which binds to oxygen
The order of amino acids alone does not always provide enough information for a protein to function. Describe how a tightly bound small molecule can influence protein function.
Small, nonprotein molecules help proteins perform functions that would be difficult or impossible with just amino acids. For example, the molecule hemoglobin is made of 4 polypeptide chains that have a heme moiety, which allows hemoglobin to pick up oxygen and carry it around the blood.
Amino acids are taken up by animal cells using a symport in the plasma membrane. What is the most likely ion whose electrochemical gradient drives the import? Is ATP consumed in the process?
Sodium. ATP is needed to drive the Na+ pump to fuel the gradient
List a few different functions of secreted proteins
Some are incorporated into the extracellular matrix, others diffuse into the ECF to nourish or signal other cells, hormones, mucus, digestive enzymes, some are attached to the cell surface
Different organelles are composed of different proteins. Some proteins enter organelles directly from the cytosol and others are delivered via membrane-enclosed compartments, or transport vesicles. Name two organelles that contain proteins that were not synthesized on free ribosomes in the cytosol.
Some mitochondrial and chloroplast proteins were synthesized on ribosomes inside the organelles
Four methods to break open cells:
Sonication Detergent Mechanical force to shear cells Cells are forced through a small hole using high pressure to break open cells
What protein model would you use if you want to show what lysozyme looks like with IVY2 bound?
Space-filling model because this shows the surface of the protein and when things bind to proteins they usually bind into a cavity on the surface
How can active transport occur across a plasma membrane?
Special transport proteins called pumps
________________ determine the orientation of transmembrane proteins
Start and stop signals
Would you expect the tripeptide with the amino acid sequence -serine-threonine-tyrosine- to be found on the inner core or the surface of a cytosolic protein?
Surface
A particular type of Drosophila mutant becomes paralyzed when the temperature is raised, because the temperature affects the structure of dynamin, causing it to be inactivated at higher temperatures. The paralysis suggests that at the elevated temperature, synaptic transmission between nerve and muscle cells is blocked. Why would signals at a synapse require dynamin? What would you see under a microscope of these flies?
Synaptic transmission involves the release of neurotransmitters by exocytosis, and during this event, the membrane of the synaptic vesicle fuses with the plasma membrane of the nerve terminals. To make new synaptic vesicles, membrane must be retried from the plasma membrane by endocytosis. This endocytosis step is blocked if dynamin is defective. On a microscope, there will be many invaginations of the membrane, representing clathrin-coated pits that cannot pinch off
What level of protein organization is it when an amino acid that is positively charged interacting with a negatively charged amino acid that folds it into a 3D structure?
Tertiary because
Phospholipid bilayers form sealed spherical vesicles in water. assume you have constructed lipid vesicles that contain Na+-K+ pumps as the sole membrane protein, and assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle. all the Na+-K+ pumps have the portion of the molecule that normally faces the cytosol oriented toward the outside of the vesicles. What would happen if you add ATP and your vesicles were suspended in a solution containing both Na+ and K+ ions and had a solution with the same ionic composition inside them.
The ATP becomes hydrolyzed, and Na+ is pumped into the vesicles, generating a concentration gradient of Na+ across the membrane. at the same time, K+ is pumped out of the vesicles, generating a concentration gradient K+ of opposite polarity. when all the K+ had been pumped out of the vesicle or the ATP ran out, the pump would stop.
If a transmembrane protein has an internal start transfer sequence, but no stop transfer sequence, what end of the protein is in the cytosol, and why is this?
The N terminus will be in the cytosol. The protein started to be made in the cytosol, and the N-terminus was the first part to be made, and when it synthesized the start-transfer signal, the SRP saw it, blocked translation, brought it to the ER membrane and everything that got translated downstream of the signal sequence was placed into the ER
What common feature of alpha helices and beta sheets makes them universal building blocks for proteins?
The N-H and C=O groups in the backbone are engaged in hydrogen bonds to give stability to the secondary structure elements and allows them to form in different proteins
Predict the membrane orientation of a protein synthesized with an uncleared internal signal sequence but that has no stop-transfer sequence.
The N-terminus will be in the cytosol, and the C-terminus will be in the ER lumen and it will pass through the membrane one time. • The protein started to be made in the cytosol, and the N-terminus was the first part to be made, and when it synthesized the start-transfer signal, the SRP saw it, blocked translation, brought it to the ER membrane and everything that got translated downstream of the signal sequence was placed into the ER
When too many unfolded proteins accumulate in the ER the unfolded protein response (URP) is triggered. Describe the role of the URP in adult-onset diabetes
The URP is when the "quality control system" is overwhelmed and the chaperone proteins cannot bind to anymore and the misfolded proteins accumulate in the ER, prompting the cell to produce more ER and chaperone proteins for quality control, but sometimes the expanded ER cannot cope and the UPR directs the cell to undergo apoptosis. In adult-onset diabetes, tissues become resistant to the effects of insulin, and to compensate, the insulin-secreting cells in the pancreas produce more and more insulin until their ER reaches maximum capacity and the UPR triggers cell death. As more insulin-secreting cells are elimated, the demand on the surviving cells increases, increasing the chance that they will die too.
Describe how clathrin together with the proteins adaptin and dynamin facilitate the transport of cargo molecules from the plasma membrane inward.
The assembly of the coat helps drive the budding process and the incorporation of cargo receptors + their cargo to the surface. On the cytosolic side of the plasma membrane, clathrin & adaptin molecules assemble and start to shape the membrane into a vesicle. Cargo molecules are bound to cargo receptors, which are captured by adaptins. Adaptins are also important to secure the clathrin coat to the vesicle membrane. A GTP-binding protein called dynamin assembles as a ring around the neck of each invaginated pit and causes the ring to constrict and pinch off the parental membrane when they hydrolyze their bound GTP. After budding is complete, the coat proteins are removed and the naked vesicle can fuse with its target membrane.
How is it possible for a change in a single amino acid in a string of 1000 amino acids to destroy its function, even when that amino acid is far away from the ligand binding site?
The atoms at the binding sites of proteins must be precisely located to fit the molecules that they bind. Their location in turn requires the precise positioning of many of the amino acids and their side chains in the core of the protein. Even a slight change in the core can disrupt protein function by altering a binding site far away
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipids tails were saturated?
The bilayer would be very viscous
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipids tails were all unsaturated?
The bilayers would be very fluid and not pack well. There would be more gaps and the bilayer would be more permeable
What happens to misfolded proteins in the ER?
The chaperone proteins within the ER hold them until they fold properly so they do not aggregate within the cell, and if they do not ever fold properly, they are degraded
Name the two characteristics of hydrocarbon tails that increase fluidity of the bilayer.
The closer and more regular the packing of the hydrocarbon tails, the more viscous and less fluid the bilayer will be. Length is important, because the shorter the chain length, the less the hydrocarbon tails will interact with each other, increasing the fluidity and the number of double bonds they contain is important because one hydrocarbon tail tends to have one or more double bonds between adjacent carbon atoms and a second tail has only single bonds between carbon atoms, and the chain that has the double bond does not have the maximum number of hydrogens attached to its carbon backbone and is unsaturated. Each double bond in the tail makes a kink, which makes it more difficult for the tails to pack against one another. The more double bonds, the more fluid the bilayer is
Which cellular component both holds organelles in place and moves them around the cell?
The cytoskeleton, especially microtubules
What is the endosymbiotic theory?
The endosymbiotic theory explains the origin of the mitochondria and chloroplast and says how the eukaryotic cell came to be. It says mitochondria and chloroplasts in eukaryotic cells were once independent prokaryotic cells, and a long time ago, there were three cells, one that was capable of aerobic respiration and converting energy, one that was capable of photosynthesis and one that was incapable of doing either of these processes, and engulfed the photosynthetic and respiratory cell. Once they were engulfed, the eukaryotic cell was now able to make useful energy and the chloroplast and mitochondria had a safe place to live and had all of the nutrients that the cytoplasm provided. Because not all eukaryotic cells have chloroplasts, but they all have mitochondria, it is thought that the pre-eukaryote engulfed the mitochondria first, and then the chloroplast.
What is the trans face of the Golgi?
The exit face of the Golgi that faces towards the plasma membrane; proteins exit from the trans Golgi in vesicles destined for the cell surface or another organelle
Compare the hydrophobic forces that hold a membrane protein in the lipid bilayer with those that help proteins fold into unique 3D structures
The exposure of a hydrophobic amino acid side chain to water is energetically unfavorable and there are 2 ways that these side cashing can be sequestered away from water. they can form transmembrane segments that span the lipid bilayer, which requires them to be in a sequence on a polypeptide chain and secondly the hydrophobic amino acid side chains can be sequestered in the interior of the folded polypeptide chain, which is one of the major forces that lock the polypeptide chain into its structure
What is the cis face of the Golgi?
The face that faces the ER and where transport vesicles from the ER dock
How can the lipid bilayer be fluid, yet asymmetrical?
The fluidity of the bilayer is strictly confined to one plane, they can diffuse laterally, but do not readily flip from one layer to the other, unless transferred by an enzyme. After proteins are inserted into the membrane in the ER, they are scrambled by scramblases to make them random
To carry out its function, a protein must be in what structure?
The folded tertiary structure aka its full 3D conformation
Describe the type of bonds that form and identify the atoms involved in the formation of a-helices and b-sheets form in proteins.
The formation is a result of hydrogen bonds that form between N-H and C=O groups in the polypeptide backbone and the protein chain adopts a regular, repeating form. The a helix is generated when a polypeptide chain turns around to make a cylinder and a hydrogen bond is made between every 4th amino acid, linking the C=O of one peptide bond to the N-H of another. A b sheet is made when hydrogen bonds form between segments of a polypeptide chain that lie side by side to produce a rigid pleated structure
How are proteins able to move through a gel?
The gel is full of a labyrinth of tunnels
An enzyme isolated from a mutant bacterium grown at 20˚C works in a test tube at 20˚C, but not at 37˚C where this bacterium normally lives. Furthermore, once the enzyme has been exposed to the higher temperature, it no longer works at the lower one. The same enzyme isolated from the normal bacterium works at both temperatures. Why?
The heat-inactivation of the enzyme suggests that a mutation cuases the enzyme to have a less stable structure. Maybe a hydrogen bond that is normally formed between 2 amino acid side chains might no longer be formed because the mutation replaces one of these amino acids with a different one that cannot participate in the bond. Lacking the bond that normally helps keep the polypeptide chain folded properly, the protein unfolds at high temperatures. Polypeptide chains that denature when the temperature is raised aggregate and rarely refold into active proteins when the temperature is decreased
Consider a transmembrane protein complex that forms a hydrophilic pore across the plasma membrane of a eukaryotic cell allowing Na+ to enter. The pore is made of five similar protein subunits, each of which contributes of a membrane-spanning alpha helix to form the pore. Propose a possible arrangement of these five alpha helices in the membrane and distribution of hydrophilic and hydrophobic amino acid side chains on each alpha helix.
The hydrophilic faces of the membrane spanning alpha helices, each contributed by a different subunit, would come together to form a pore that is lined with the hydrophilic amino acid side chains so ions can pass without coming in contact with the bilayer
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipids tails were shorter than normal?
The lipid bilayer would be much more fluid because the tails would interact less and the bilayers would be less stable, as the shorter hydrocarbon tails would be less hydrophobic and the forces to drive the formation of the bilayer would be reduced
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipids tails were covalently linked to the tail of the other lipid on the opposite monolayer?
The lipid bilayers formed would have unchangeable properties because if they were linked they would not be able to diffuse within their monolayer or rotate and the membrane would not be as fluid
Do lipid molecules move in the membrane or are they fixed in place? Explain.
The membrane is fluid and flexible. The phospholipids can exchange places with each other, and they flex their hydrocarbon tails and rotate rapidly on their long axis
Some E. coli bacteria are living within the unpasteurized milk in your fridge. You drink the milk, and the bacteria is now inside your warm human body. What sort of changes might occur in order for the E. coli to maintain its ideal membrane fluidity?
The membrane is now in a warm environment, and in order to counteract this, they need to decrease the fluidity of their membrane, so they would increase their tails, have more saturated to unsaturated tails, and increase cholesterol
Acetylcholine-gated cation channels do not discriminate between Na+, K+, and Ca2+ ions, allowing all to pass freely. So why is it that when ACh binds to this protein in the membrane of muscle cells, the channel opens and there is a large influx of primarily Na+?
The membrane potential and steep extracellular Na+ concentration provide a large inward electrochemical driving force. Ca2+ will enter, but they are limited by their limited extracellular concentration and K+ will barely enter since there is a high intracellular concentration
What would be the result if the amount of cholesterol in the membrane was doubled?
The membrane would become stiff and would lose a large amount of its fluidity because cholesterol fills the spaces between phospholipids
What is meant by the term 2D fluid?
The molecules are free to move only in 1 plane
Predict how membrane fluidity will affect the mobility of integral membrane proteins
The more fluid the membrane is, the more the proteins can move around in the membrane because integral membrane proteins are linked to lipids, and the more fluid the membrane is, the more that lipids can move around
What would happen if unbound phospholipids were placed in a non polar solution?
The non polar tails would be out facing the solution, and the polar heads would cluster together and try to prevent exposure to the non polar solution
Protein stability is influenced by what?
The number and the strength of the bonds that form between amino acids in the polypeptide chain influence protein stability.
What type of proteins are synthesized on the rough ER?
The ones that go to the Golgi and get exported or are targeted for certain organelles
Which of the following amino acids would you expect to find more often new the center of a folded globular protein? Which ones will be exposed on the outside? Ser Ser-P Leu Lys Gln His Phe Val Ile Met Cys-S-S-Cys Glu
The polar amino acids, Ser, Ser-P, Lys, Fan, His, and Glu will be found on the surface and the hydrophobic amino acids Leu, Phe, Val, Ile, and Met will be fond on the interior. The oxidation of the 2 cysteine residues to form the disulfide bond eliminates the potential for them to form hydrogen bonds, so they will be hydrophobic and on the interior of the protein. The N terminal and C terminal amino acids contain charged groups and are found on the protein surface
What interacts to fold proteins?
The polar side chains (the polar side chains will want to interact and the nonpolar side chains will want to be tucked away)
How does the amino acid sequence relate to a protein's identity?
The position of each amino acid determines its 3D shape because the amino acids are differently charged and some are polar and some are nonpolar, and noncovalent bonds between amino acid side chains form and also becasue the nonpolar side chains will want to be onthe inside of the protein becasue the cell is an aqueous environment, and the polar will be on the outside, so they will fold in a way that reflects that and a protein's function is determined by its shape
What 3 things does a Western blot detect?
The presence, abundance, and size of proteins
How is a primary antibody different than a secondary antibody?
The primary antibody directly binds to the target protein, but it is not a reporter, it is just for the proper binding. The secondary antibody is the reporter antibody that binds to the primary antibody and the secondary antibody is also attached to an enzyme, and once a substrate is added, a product appears that is colored
Many ER localized transmembrane proteins are composed of hydrophobic start-and stop transfer sequences. How do start and stop signals determine the arrangement of transmembrane proteins in the lipid bilayer?
The process starts out like it does for soluble proteins, where the signal sequence on the N-terminus initiates translocation, but then the transfer process is halted by a hydrophobic sequence of amino acids called a stop-transfer sequence and the translocation channel releases the growing polypeptide chain into the lipid bilayer, and the signal sequence gets cleaved off. The stop-transfer sequence forms an alpha-helical membrane spanning segment that hoods the protein in the membrane. Once inserted into the membrane, a transmembrane protein does not change its orientation, which is retained throughout any budding or fusion events. In transmembrane proteins that pass back and forth across the bilayer, there is a start transfer signal that is used as a signal sequence and acts to anchor the final protein in the membrane. The start transfer sequence is recognized by an SRP, which brings the ribosome to the ER membrane and the start sequence is inserted into the translocation channel and when a stop transfer sequences ir reached, the channel discharges both sequence into the lipid bilayer
Phospholipid bilayers form sealed spherical vesicles in water. assume you have constructed lipid vesicles that contain Na+-K+ pumps as the sole membrane protein, and assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle. all the Na+-K+ pumps have the portion of the molecule that normally faces the cytosol oriented toward the outside of the vesicles. What would happen if you add ATP, but the solution- outside as well as inside the vesicles contains only Na+ ions and no K+ ions
The pump would initiate a transport cycle and then cease because each reaction step must occur strictly sequentially, dephosphorylation and the accompanying conformational switch cannot occur in the absence of K. The Na+ pump will become stuck in the phosphorylated state, waiting for K+. The number of Na+ transported would be miniscule, because each pump molecule would have functioned only once.
What is a condenser on a microscope?
The purpose of the condenser lens is to focus the light onto the specimen.
What protein model would you use if you discover a mutant, nonfunctional version of lysozyme, and hypothesize that there is a missense mutation disrupting some of the protein's secondary structure, so you want to compare the structures of mutant and wildtype versions of lysozyme to see if this is correct.
The ribbon model because the ribbon model shows the secondary structure very well
Why and how is a hole in the membrane quickly and spontaneously repaired?
The same force that drives the formation of the lipid bilayer makes the bilayer self-sealing because any tear in the bilayer will create a free edge that is exposed to water, which is energetically unfavorable, so the molecules will spontaneously rearrange to eliminate the free edge. If the tear is small, the rearrangement will exclude water molecules and repair the bilayer, and if the tear is large, the sheet may fold in on itself
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipid bilayer contained a mixture of 2 kinds of phospholipid molecules, one with 2 saturated tails and one with 2 nonsaturated tails?
The saturated tails would tend to aggregate with one another because they can pack so much more tightly and would form patches of reduced fluidity. The bilayer would not be uniform. Normally, one saturated and 1 unsaturated tail are on one molecule so the bilayer stays fluid
Gel-filtration chromatography separates molecules according to their size. Smaller molecules diffuse faster in solution than larger ones, yet smaller molecules migrate more slowly through a gel filtration column than larger ones. Explain the paradox. What would happen at rapid flow rates?
The slower migration of smaller molecules through a gel-filteration column occurs because smaller molecules have acces to more spaces in the porous beads that are packed into the column. It is important to have a slow flow rate to give the smaller molcules enough time to diffuse into the spaces inside of the beads. At rapid flow rates, all molecules will move rapidly around the beads and small and large molecules will exit together from the column and the proteins would not be separated
An intestinal cell has a large concentration of potassium and a low concentration of sodium internally. It is placed in a solution rich in sodium. What will be the rate of flow and in what direction?
The sodium will flow into the cell at a fast rate because it is draw to the cell's negative internal environment as well as the low Na+ concentration in the cell. The potassium is drawn in by the cells negative potential but drawn out by the concentration gradient.
Glycolipids are unlike many other lipids found in the plasma membrane in that they specifically localize to the noncytosolic half of the bilayer. What is the significance of the localization of glycolipids to the noncytosolic half of the bilayer and why aren't these molecules ever transferred to the cytosolic half of the bilayer?
The sugar groups of the glycolipids form a continuous coat of carbohydrate that surrounds and protects animal cells and can be part of the glycocalyx. When a glycolipid is made in the Golgi, the enzymes are oriented such that sugars are added to lipid molecules on the noncytosolic half of the bilayer, so that when a glycolipid molecule is delivered to the plasma membrane, it displays its sugar to the exterior of the cell because the positioning is preserved as membranes bud from one organelle to another or to the plasma membrane. In addition, the flippases in the Golgi do not work on glycolipids
Why can't most eukaryotic cells be used to produce and accumulate genetically coupled immunotoxins?
The toxin will inhibit the host-cell proliferation
There are many vesicles being transported around the cell at any given time, many of which have different destinations. How would a vesicle carrying proteins for a specific organelle be recognized but that organelle but not others?
There are specific proteins called Rab proteins on the coat of transport vesicles that identify the vesicle according to its origin and cargo that are recognized by complementary receptors, called tethering proteins, on the appropriate target membrane. Specific Lab protein on the surface of each type of vesicle are recognized by corresponding tethering proteins on the cytosolic surface of the target membrane and each organelle and each type of transport protein contains a unique combination of Rab proteins
How is the transport of glucose against its concentration gradient facilitated by the electrochemical gradient of Na+?
There is a symport in the apical surface of the gut epithelial cells that relies on the electrochemical gradient of sodium that wants to move into the cell to "drag" glucose along with it because in order for the pump to close and shift to the other side of the cell, it must have both a sodium and a glucose molecule bound to it.
Aquaporins are transmembrane proteins which enable water to be transported into and out of the cell. Mutations in mice aquaporin have been linked to cataracts. What structural changes may cause the protein to become dysfunctional?
These are going to be integral proteins that go from the aqueous environment, though the hydrophobic interior of the membrane, to the cytosol. The nonpolar sidechains in the membrane are exposed to the nonpolar tails. If a mutation caused these nonpolar regions to become polar, the protein would lose its shape and no longer be able to function. Changing a polar group exposed to either polar side to a nonpolar group might also cause such a folding.
GTP-binding proteins regulate protein function in eukaryotes how?
They act as molecular switches that are active when GTP is bound and inactive when GDP is bound; turning themselves off by hydrolyzing their bound GTP to GDP. They can also control other proteins by binding them to control their activities and control downstream targets of signaling cascades
What is a similarity and difference between membrane potential and electrochemical gradient?
They are both describing gradients across a membrane, but membrane potential is voltage gradient and electrochemical gradient is both voltage and concentration gradient of a particular solute. Membrane potential is defined independently of a solute.
Why are detergent molecules more water-soluble than phospholipids and what is the implication of this?
They are more water soluble since they have shorter hydrocarbon tails, and this is important so they can leave and re-enter micelles in aqueous solution and also form monomers to enter the lipid bilayer and solubilize the proteins
Proteins moving from the cytosol into the nucleus enter through a nuclear pore whereas proteins moving from the cytosol in the ER, mitochondria, or chloroplast enter through protein translocators located in the membrane. How does protein transport via the endomembrane system differ?
They are transported by transport vesicles that pinch off from the membrane of one compartment and then fuse with the membrane of another. The transport vesicles deliver soluble cargo proteins, as well as the proteins and lipids that are part of the vesicle membrane
What is a similarity and difference between a pump and a transporter?
They both transport molecules across the membrane and are membrane proteins, but a pump uses energy to transport a solute uphill against an electrochemical gradient if the solute is charged, or a concentration for a charged solute
How do reducing agents affect a protein structure?
They break the disulfide bonds
There are 3 different tools that a researcher could use to visualize neurons in a mouse's brain that fluoresce! To obtain the image, what could they use?
They could express a transgene that was: Promoter of neuron gene + coding region of neuron gene + GFP They could use a fluorescent microscope if the neurons were stained with fluorescent dyes. Use antibodies conjugated to a fluorophore that recognize a protein that is abundant in and localized to neurons
Can non polar amino acids participate in bonding that helps determine the shape of the protein?
They do not necessarily bond, but they interact and find energetically favorable locations that contributes to protein shape
In the ER lumen, what happens to proteins?
They fold up, assemble with their protein partners, form disulfide bonds, and become decorated with oligosaccharide chains
Identify a couple of similarities between prokaryotes and mitochondria and chloroplast.
They have their own genetic material, DNA in a circular chromosome Have their own ribosomes Replicate independently by binary fission
What are the reasons why many vesicles are coated?
They help shape the membrane into a bud and they capture molecules for onward transport
Why are intrinsically disordered sequences in proteins important?
They help the protein maintain flexibility to be able to fold into a structure. Protein structures are not fixed and these regions allow proteins to be dynamic
A signal sequence can be both "necessary and sufficient" for protein localization. Hypothetically, if a certain signal sequence is necessary, but not sufficient for protein localization, what would this mean?
This means that if you have the signal sequence to localize the protein, you still need something else in order for the protein to localize to its correct place. -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val- This is a signal sequence that takes a protein to the nucleus normally. For example, if you mutate one of the bolded lysine, and it doesn't go to the nucleus, you know lysine is necessary, but if you put the lysine back in, and take out the valine, you know that lysine is not sufficient and you also need the valine
How would losing the function of the Na+/K+ transporter alter a Na+/glucose transporter in the same cell?
This pump is necessary to remove Na+ from the cell to keep its intracellular concentration low, and so if this pump does not work, then the intracellular sodium concentration would be higher and the drive of sodium to get into the cell would be lower and also because the sodium-potassium pump removes 3 x Na+ for ever 2 x K+ it puts in, it helps keep the cell more negative than the outside of the cell
If membrane proteins are integrated into the ER membrane by means of the ER protein translocator, which is a membrane protein, how do the first protein translocation channels get into the ER membrane?
This situation never arises today because new cell membranes are made by the expansion of old cell membranes and so the ER is not made de novo. Inheritance is not limited to the genome, a cell's organelles are passed from generation to generation too
The structure of the lipid bilayer is determined by the properties of the lipid molecules. What would happen if phospholipids had only 1 hydrocarbon tail instead of 2?
This would be like detergent. The diameter of the lipid head would be much larger than that of the hydrocarbon tail, so the shape of a molecule would be a cone and the molecules would aggregate to form micelles rather than bilayers
What is the role of a detergent and centrifugation in the first step of a western blot?
To break down the cell membrane and the cytoplasm contents can come out, and the centrifuge gives the supernatant and a pellet, with the pellet containing the cell fragments and cellular debris and the supernatant containing all of the proteins
Why might a scientist use western blotting.
To detect proteins present in a mixture
You have successfully identified a gene required for cell division in yeast called Cdc2. Yeast with loss-of-function mutations in Cdc2 cannot divide at warm temperatures. You are particularly interested in the protein encoded by Cdc2 because your data suggests that the causative mutation does not affect gene expression. In other words, you observed no difference in the relative abundance of RNA transcript in the mutant. What is a protocol that could test this hypothesis: The protein is targeted for degradation in the mutant and thus is less abundant or even absent in mutant yeast.
To determine if the protein is targeted for degradation in the mutant and less abundant, we will run a western blot. 1) I will grow 2 plates of cells in an incubator, one mutant yeast grown on plates with growth media and one wildtype yeast grown IN AN INCUBATOR SINCE THE MUTANT IS TEMPERATURE SENSITIVE 2) We will split open the cells using sonication to create a homogenate 3) We will centrifuge the homogenate to separate the pellet and the supernatant 4) We will use pipettes to remove the supernatant and heat the supernatant with SDS and mercapoethanol to denature the proteins and make them negatively charged. It is important to denature the proteins so that they travel through the gel based on size, and their folding does not impact this 5) We will load the total protein into the polyacrylamide gel and run the gel (Lane 1: ladder; Lane 2: WT; Lane 3: Mutant) 6) After the run, we will transfer the proteins from the gel to the nitrocellulose paper 7) We will use milk to prevent any nonspecific blocking 8) After the transfer, we will use a primary antibody that recognizes and binds to the proteins (they will bind to both the wildtype and the mutant because the antibody recognizes such a small part of the protein that it probably will not differentiate between the wildtype and mutant that were made in a rabbit, and a secondary anti-rabbit antibody that is tagged with a red fluorophore 9) Develop the film 10) After developing the film, I will look to see if there are thicker bands for the wildtype, suggesting there is more protein in the wildtype than the mutant
What is the purpose of PCR?
To make many copies of a specific region of DNA
Iron (Fe) is an essential trace metal that is needed by all cells. it is required, for example, for synthesis of the heme groups and iron-sulfur centers that are part of the active site of many proteins involved in electron-transfer reactions; it is also required in hemoglobin, the main protein in red blood cells. Iron is taken up by cells by receptor-mediated endocytosis. the iron-uptake system has two components: a soluble protein called transferrin, which circulates in the bloodstream; and a transferrin receptor—a transmembrane protein that, like the ldl receptor, is continually endocytosed and recycled to the plasma membrane. Fe ions bind to transferrin at neutral pH but not at acidic pH. transferrin binds to the transferrin receptor at neutral pH only when it has an Fe ion bound, but it binds to the receptor at acidic pH even in the absence of bound iron. From these properties, describe how iron is taken up, and discuss the advantages of this elaborate scheme.
Transferrin without Fe bound does not interact with its receptor and circulates in the blood until it catches an Fe. Once iron is bound, the complex can bind to the transferrin receptor on the surface of the cell and be endocytosed. Under acidic conditions of the endoscope, the transferrin releases its iron, but transferring remains bound to its receptor, which is recycled to the cell surface, where it encounters a neutral pH environment of the blood. The neutral pH causes the receptor to releases transferrin to pick up more Fe to repeat. The iron released in the endosome moves to the lysosome and to the cytosol. This system allows cells to take up iron efficiently
Differentiate between transporters, channels, and ion channels
Transporters shift small organic molecules or inorganic ions from one side of the membrane to another by changing shape. Transporters are very specific with what they transport and they bind to their molecule very tightly. Channels form hydrophilic pores across the membrane through which substances can pass by diffusion and do not undergo a conformational change and ion channels are channels that permit only inorganic ions to pass. Channels only participate in passive transport, while transport proteins participate in active or passive transport
What are the different functions of plasma membrane proteins?
Transporters, channels, anchors, receptors, enzymes
T/F, Most molecules cannot simply diffuse across the plasma membrane
True
T/F: Certain H+ pumps are fueled by light energy
True
T/F: Ribosomes are cytoplasmic structures that, during protein synthesis, become linked by an mRNA to form polyribosomes
True
T/F: The Na+/K+ pump moves 3 Na+ ions out of the cell for ever 2 K+ ions it moves into the cell
True
T/F: Transport vesicles deliver proteins and lipids to the cell surface
True
True or False? All of the proteins on an SDS-PAGE gel are transferred to nitrocellulose paper when one is performing a western blot.
True
True or False? One third of all highly structured eukaryotic proteins contain disordered regions.
True
True or false, glycolipids move between different membrane enclosed compartments during their synthesis, but remain restricted to one side of the bilayer
True
True or false, lipids in a lipid bilayer do not flip flop readily from one monolayer to the other
True
True or false, some membrane proteins are enzymes
True
True or false, allosteric enzymes have 2 or more binding sites
True (allosteric enzymes have a binding site for the substrate and a regulatory binding site and because enzymes are very, very specific, the chances that the regulatory molecule and substrate are shaped identically is very unlikely and these sites "communicate" via allosteric changes)
True or false, catalysis by some enzymes involves the formation of a covalent bond between an amino acid side chain and a substrate molecule
True, but in all cases, the enzyme is restored to its original structure after the reaction
T/F: All transport vesicles in the cell must have a v-SNARE on their membrane
True, otherwise they could not dock at the correct target membrane or recruit a fusion complex to the docking side
True or false, hydrogen bonds that form between lipid head groups and H2O molecules are continually being broken and reformed
True, the lipid bilayer is fluid because of the lipid's motions
True or false, lipids in a lipid bilayer randomly exchange positions with one another in their own monolayer
True, the lipid bilayer is fluid because of the lipid's motions
True or false, lipids randomly spin on their long axis
True, the lipid bilayer is fluid because of the lipid's motions
True or false, margarine contains more saturated lipids than the vegetable oil from which it is made
True, the reduction of double bonds allows the resulting saturated lipid molecules to pack more tightly, turning oil into margarine
T/F: If the delivery of prospective lysosomal proteins from the trans Golgi to the late endosomes were blocked, lysosomal proteins would be secreted by constitutive methods
True. (Lysosomal proteins, if not selected, enter the default transport vesicle that move to the cell surface constitutively.)
True or false, the active site of an enzyme usually occupies only a small fraction of the enzyme surface
True. Only a few amino acid side chains are contributing to the active site and the rest of the protein is required to maintain the polypeptide chain in the correct conformation, provide additional binding sites for regulatory purposes and localize the protein in the cell
T/F: The plasma membrane of many animal cells contains open K+ channels, yet the K+ concentration in the cytosol is much higher than outside the cell
True. The K+ concentration remains higher in the cell since the membrane potential is negative, this prevents more K+ from leaking out
What does heating do to proteins?
Unfolds them, but it does not break -S-S- bonds
Explain how a protein enters the ER.
Unlike most other proteins that enter the nucleus, chloroplast, mitochondria, or peroxisomes, proteins that enter the ER begin to be threaded across the ER membrane before the polypeptide chain has been completely synthesized. Signal recognition particles bind to both the ribosome and the ER signal sequence and slows the protein synthesis, so that the SRP has time to engage with an SRP receptor within the membrane of the ER. Once the SRP is bound to the SRP receptor, the SRP is released and the receptor passes the ribosome to a protein translocator that is also in the ER membrane and protein synthesis continues and the polypeptide chain is threaded across the ER membrane through a channel in the protein translocator. The signal sequence functions to open the channel of the protein translocator, and this sequence remains bound to the membrane while the rest of the protein is being threaded through. It is removed by a transmembrane signal peptidase
Why do high concentrations of urea denature proteins?
Urea is a small molecule that can donate hydrogen bonds and accept them, so it can squeeze between hydrogen bonds that stabilize proteins and destabilize protein molecules. In addition, the nonpolar side chains of a protein are held together in the interior of the folded structure because they would disrupt the structure of water if they were exposed. At high concentreations of urea, the hudrogen-bonded network of water becomes disrupted so the hydrophobic forces are diminished and proteins unfold
CFTR is a transmembrane protein that moves Cl- ions down their concentration gradient. In people with cystic fibrosis, this protein is mutated, and is often not in the plasma membrane as it should be, and instead remains in the cytoplasm. How could you test this hypothesis?
Use co-localization. Use recombinant DNA technology to conjugate the gene for GFP to the promoter and gene for protein CFTR. Introduce the transgene into wild type and mutant cells. Fix cells and then incubate with a anti-protein X primary antibody (made in mouse) against a protein known to be ubiquitous in the plasma membrane (this protein will serve as my marker) Incubate cells with a anti-mouse secondary antibody conjugated to a red fluorophore Look at cells under a fluorescent microscope. If my hypothesis is correct then I will see a red membrane around each cell and green within the cell, not at the membrane If my hypothesis is incorrect then I will see a red/green membrane (red & green overlap)
You want to test a hypothesis that a mutation prevents protein Y and protein Z from interacting. How could you do that?
Use recombinant DNA technology to conjugate one half of gene for GFP to the promoter and gene for protein Y, use recombinant DNA technology to conjugate the other half of the gene for GFP to the promoter and gene for protein Z. Introduce the transgenes into cells. Look at cells under a fluorescent microscope. If my hypothesis is correct then I will see: no fluorescence If my hypothesis is incorrect then I will see: green fluorescence
CFTR is a transmembrane protein that moves Cl- ions down their concentration gradient. In people with cystic fibrosis, this protein is mutated, and is being degraded. How could you test this hypothesis?
Use sonication to homogenize your samples of wild type and mutant cells (although keep the two samples separate). Use centrifugation to remove large, dense cell debris from the homogenate. Keep the supernatant and discard the pellet. Treat the samples with SDS in order to solubilize the proteins and coat them with a negative charge. Treat samples with mercaptoethanol in order to reduce disulfide bonds or other covalent linkages. Run samples in separate lanes of a PAGE. Transfer proteins from polyacrylamide-gel to nitrocellulose membrane. Use a blocking agent to prevent nonspecific interactions between the antibodies and the nitrocellulose paper. Incubate with a anti-protein X primary antibody (made in a donkey). Wash and then incubate with a anti-donkey secondary conjugated to a red fluorophore. Examine the membrane If my hypothesis is correct then I will see: a red band in the wild type lane and no band in the corresponding spot on the mutant lane. If my hypothesis is in correct then I will see: a red band in the wild type lane and a red band at the same position in the mutant lane.
Random mutations only very rarely result in changes in a protein that improve its usefulness for the cell, yet useful mutations are selected in evolution. Because these changes are so rare, for each useful mutation there are innumerable mutations that lead to no improvement or inactive proteins. Why do cells not contain millions of proteins that are of no use?
Useless proteins are detrimental to organisms, as they waste energy making them. If mutant proteins were made in excess, the synthesis of normal proteins would suffer and a mutant protein could interfere with the normal workings of a cell; a mutant enzyme could bind a carrier, but not catalyze a reaction, so natural selection provides a strong driving force that eliminates useless and harmful proteins
Dr. Dorian thinks that TAS2R2 relies on an allosteric activator, but does not know what the activator is. He believes that the activator is a protein in the SNF family, and has antibodies for several SNF targets. Describe at least one way to determine which protein is the allosteric activator and explain how you would read the results.
Using a double antibody labeling system, he could try using fluorescence microscopy to see if the TAS2R2 and SNF proteins co-localize within the cell.
Lost in his daydreams, Dr. Dorian accidently mixes up his TAS2R2 and several other taste receptor proteins! Describe one way that Dr. Dorian could isolate TAS2R2. How could he verify that TAS2R2 is not still in the remaining protein sample?
Using affinity chromatography. He would need to make sure that the other proteins did not also bind to cyclohexamine. Then he would run the total protein through the column, and after everything else flowed through, he could wash the column with a solvent of a different pH, and changing the pH will release TAS2R2 from the beads by altering interactions. In order to show that TAS2R2 is not in the remaining protein, a western blot could be run on the remaining protein. If no bands for the TAS2R2 show up, then it has been fully extracted. We would have a ladder in the first lane, a control in the 2nd lane with TAS2R2 and the sample
What is constitutive exocytosis?
Vesicles that are constantly budding from the trans Golgi supplying the plasma membrane with are lipids and proteins and carrying soluble proteins to the surface to be released through secretion
The influenza virus is surrounded by a membrane that contains fusion proteins which are activated by acidic pH. Upon activation, the protein causes the viral membrane to fuse with cell membranes. An old folk remedy against the flu recommends that one should spend the night in a horse stable because the air contains NH3. How would this protect cells from viral infection?
Viruses enter cells by endocytosis, and things that are endocytosed go to endosomes where it encounters an acidic pH that activates its fusion protein, which binds to the membrane of the endoscope and releases the viral genome into the cytosol. NH3 is small and can penetrate the cell membrane and can also enter all intracellular compartments by diffusion. In the endosome, NH3 binds with H+ to form NH4+, which is charged and cannot cross the membrane, so NH4+ accumulates in the endosome and raises the pH and when the pH of the endosome is raised, the influenza virus cannot fuse and enter the cytosol and will end up in the lysosome
What are the three ways that proteins are transported into organelles?
Water soluble proteins face a problem getting through the membrane of organelles, because the membranes are usually impermeable to hydrophilic macromolecules. Proteins move from the cytosol into the nucleus through nuclear pores. Proteins move from the cytosol to the ER, mitochondria, and chloroplasts by protein translocators in the membrane. Proteins moving onward from the ER to other parts of the endomembrane system are transported by transport vesicles
Do we find larger proteins on the reduced or non-reduced Western blots?
We find larger proteins under non-reducing conditions, meaning that in the non-reduced conditions there were disulfide bonds that kept them folded so they could not travel as far, or because the disulfide bond was holding together 2 polypeptide chains to create a larger protein. Under reducing conditions, we will only have proteins migrate at a rate that affects their molecular weight, not at a rate that affects their molecular weight and folding
Coiled coils form when a-helices wrap around one another. Why would a coiled coil form?
When a helices have most of their nonpolar side chains on one side so they can twist around each other with the side chains facing inward to minimize contact with the cytosol
Detergents are used in the laboratory to lyse cells during protocols that require DNA and protein extraction. How do detergents lyse a cell?
When detergent is mixed with membrane, the hydrophobic ends of the detergent molecules interact with the hydrophobic regions of the transmembrane proteins and the hydrophobic tails of phospholipids, disrupting the lipid bilayer and separating proteins from phospholipids. Normally, phospholipids would spontaneously reassemble a bilayer because it is the most energetically favorable, but when the detergent molecules attach to the phospholipids, they form a sealed space and prevent the membrane from forming
What is active transport?
When molecules cannot cross the membrane without use of a protein to transport them and they go up their concentration gradient and require energy to cross the membrane. Energy can come in the form of ATP, light, or another ion gradient (coupled transport)
Why does heat denature proteins?
When the heat rises, so does the kinetic energy, so the atoms move around and break the weak hydrogen bonds
Why is a nitrocellulose membrane used in western blotting?
When we use a probe, the probe can bind to different regions of the gel, but gel is really fragile and the probing and the reactions necessary cannot take place on the gel and it has a high affinity for proteins
Golgi
Where proteins are packaged and moved into vesicles to be transported out of the cell or to other places in the cell and it is where proteins get folded and modified
For neurons, how does cell shape fit function?
With the long axon, they are able to transmit electrical signals far distances With the many dendrites, they can receive signals from many different other neurons
The budding of clathrin-coated vesicles from eukaryotic plasma membrane fragments can be observed with adaptins, clathrin, and dynamin-GTP are added to the membrane preparation. What would you observe if you omitted adaptins?
Without adaptin, the clathrin cannot attach to the membrane and the receptor and its cargo cannot bind either
The budding of clathrin-coated vesicles from eukaryotic plasma membrane fragments can be observed with adaptins, clathrin, and dynamin-GTP are added to the membrane preparation. What would you observe if you omitted dynamin?
Without dynamin, deeply invaginated pits form, but they do not pinch off
Can H2O cross the plasma membrane freely?
Yes
Can O2 cross the plasma membrane freely?
Yes, small and non polar
Can CO2 cross the plasma membrane freely?
Yes, small and nonpolar
Can steroids cross the plasma membrane freely?
Yes, small and nonpolar
Can N2 freely cross the plasma membrane? Why or why not?
Yes. It is small, not charged and non polar
What is facilitated diffusion?
a form of passive transport that utilizes transport proteins to move solutes across the membrane and does not require chemical energy
Chromatography
a matrix of small beads that separates proteins according to charge, size, hydrophobicity, or ability to bind to a specific molecule.
Renaturation
a protein spontaneously folding back into its original conformation after denaturation. Not many proteins can do this, but under the right conditions, some can and this proves that all of the information necessary to specify the 3D shape of the protein are contained in its amino acid sequence
In affinity chromatography, what can we use to release a molecule bound to the column?
a solvent that alters the pH of the column to change the shape of the protein
In gel filtration chromatography, what can we use to release a molecule bound to the column?
a solvent that washes the molecule out of the pores
Large protein molecules often contain more than 1 polypeptide chain, and each polypeptide chain in such a protein is called ________-
a subunit
For epithelial cells, how does cell shape fit function?
aWith the microvilli, they are able to increase surface area to absorb more nutrients
Microfilaments
allow cytoplasm to move because the actin filaments rapidly assemble and disassemble to casue motion
All the information required to determine a protein's conformation is contained in its ___________ .
amino acid sequence
Membrane-associated proteins do not span the bilayer and instead associate with the membrane through an α helix that is___________, meaning it possesses both hydrophilic and hydrophobic properties.
amphipathic
The ligand of an antibody is known as
an antigen
Monolayer-associated alpha helix protein
anchored to the cytosolic half of the lipid bilayer by an amphipathic alpha helix
Micelles form spontaneously in ________
aqueous solution
Lipid linked protein
attached to either side of the bilayer solely by a covalently attached lipid molecule
Protein attached membrane protein
attached to the membrane by relatively weak, noncovalent interactions with other membrane proteins
Why does H2O often travel through the plasma membrane in aqua porins if it can freely flow through?
because often the rate that the cell need's water is faster at which it can get through because it is polar and doesn't cross super fast, but it can freely cross
Nucleus
carries all of the genetic information of the eukaryote
Mitochondria
carry the final oxidation of food molecules and generate ATP
All of the proteins (what scientists call the "total protein") in cells that were homogenized are loaded into an SDS-PAGE gel. Although the proteins are all the same_______ (charge/size) they differ in _______ (charge/size) and thus move through the gel at different rates.
charge; size
Covalent bond
chemical bond that involves the sharing of electron pairs between atoms.
Noncovalent bond
chemical bond that typically bond between macromolecules. They do not involve sharing a pair of electrons. Noncovalent bonds are used to bond large molecules such as proteins and nucleic acids.
What type of DNA do prokaryotes have?
circular
What type of DNA does a chloroplast have?
circular chromosome
What type of DNA does a mitochondrion have?
circular chromosome
Order these by size: adenine, x-chromosome, phage, tRNA, sperm, coffee bean, sesame seed, mitochondria, E.coli bacterium
coffee bean, sesame seed, sperm, X-chromosome, mitochondria, E. coli, phage, tRNA, adenine
In a SE microscope, what is the component that promotes an interaction between the sample and the energy?
condenser
In a TE microscope, what is the component that promotes an interaction between the sample and the energy?
condenser
Lipids and proteins travel to the plasma membrane in vesicles that fuse with the plasma membrane via a process called
constitutive exocytosis
Cytoplasm
contains all of the cell's contents outside of the nucleus and contains membrane enclosed organelles
Ligand-gated channels are controlled by what?
controlled by binding a ligand to the channel
Mechanically-gated channels are controlled by what?
controlled by mechanical force applied to the channel
Voltage-gated channels are controlled by what?
controlled by the membrane potential
Some proteins are strongly, or ____________ attached to lipid molecules that are inserted in the membrane. ___________ membrane proteins however are linked to the membrane through noncovalent interactions with other membrane-bound proteins.
covalently; peripheral
Interior of the nuclei get their proteins from where?
cytosol
Chloroplasts get their proteins from where?
cytosol, since they are not part of the endomembrane system
The _______ makes up about half of the total cell volume of a typical eukaryotic cell. Ingested materials within the cell will pass through a series of compartments called _________ on their way to the ________ , which contains digestive enzymes and will ultimately degrade the particles and macromolecules taken into the cell and will also degrade worn-out organelles. The _________ has a cis and trans face and receives proteins and lipids from the ________ , a system of interconnected sacs and tubes of membranes that typically extends throughout the cell.
cytosol; endosomes; lysosome; golgi; ER
On being heated, a protein molecule will become ________as a result of breakage of ________ bonds.
denatured; noncovalent
Lysosomes
digest large molecules and contain digestive enzymes. They release nutrients from digested food and break down unwanted particles to recycle or for secretion
Proteins found in thermophilic organisms will probably have proteins with what in them?
disulfide bonds
A protein __________ is the modular unit from which many larger single-chain proteins are constructed.
domain
What is the source of energy to interact with the specimen for the scanning electron microscope?
electrons
What is the source of energy to interact with the specimen for the transmission microscope?
electrons
The ribbon model of proteins shows what?
emphasizes and shows folds, secondary structure is easy to see with the ribbon model
Eukaryotic cells are continually taking up materials from the extracellular space by the process of _________. One type of endocytosis is pinocytosis , which uses __________ proteins to form small vesicles containing fluids and molecules. After these vesicles have pinched off from the plasma membrane, they will fuse with the __________ , where materials that are taken into the vesicle are sorted. A second type of endocytosis is _________ , which is used to take up large vesicles that can contain microorganisms and cellular debris. Macrophages are especially suited for this process, as they extend (sheetlike projections of their plasma membrane) to surround the invading microorganisms.
endocytosis; clathrin; endosome; phagocytosis
A lot of chemical substances in our bodies cannot cross membranes, and we have to transport those via the
endomembrane system
Transmembrane protein
extend across the lipid bilayer as a single alpha helix, multiple alpha helices, or as a rolled up beta sheet
True or false, noncovalent bonds are too weak to influence the 3D protein structure
false, single noncovalent bonds are weak, but many bonds act together to contribute to the 3D structure
Cytosol
fluid portion of the cytoplasm
Microtubules
form mitotic spindles and make up the cilia and flagella
Cytoskeleton
forms the framework of the cell and cosnists of microfilaments, microtubules, and intermediate filaments that continually reconstruct to adapt the cell's changing needs
Bind to DNA to switch genes on or off
gene regulatory proteins
What is an ATP-driven pump?
hydrolyzes ATP to drive uphill transport. The energy from ATP hydrolysis induces a series of protein conformational changes and as part of the process, the phosphate removed from ATP gets transferred to the pump to change its shape
How does membrane composition vary between different organelles?
iDifferent organelles have different lipid composition and as a result different proteins can associate with the membranes to support different functions
What is immunostaining?
immunostaining is any use of an antibody-based method to detect a specific protein in a sample. (i.e. Western blot)
Golgi get their proteins from where?
indirectly from the ER
Inner nuclear membranes get their proteins from where?
indirectly from the ER
Lysosomes get their proteins from where?
indirectly from the ER because it is part of the endomembrane system, but they also get proteins from proteins that have been endocytosed via endosomes
Endosomes get their proteins from where?
indirectly from the ER via the Golgi and also through endocytosis
What are the different types of beads that can be used in column chromatography?
ion-exchange, gel-filtration, and affinity
Lipid composition varies between different ___________ of a membrane.
leaflets
What is the source of energy to interact with the specimen for the florescent microscope?
light
What type of organelles do prokaryotes have?
little to none
What is secondary protein structure?
localized folding that is stabilized by hydrogen bonds; includes alpha-helices and beta-sheets
Endosomes mature into _______________.
lysosomes
What type of organelles do eukaryotes have?
many, mostly membrane bound
Cells can be very diverse: superficially, they come in various sizes, ranging from bacterial cells such as Lactobacillus, which is a few _____________ in length, to larger cells such as a frog's egg, which has a diameter of about one__________. Despite the diversity, cells resemble each other to an astonishing degree in their chemistry. For example, the same 20 ____________ are used to make proteins. Similarly, the genetic information of all cells is stored in their_________ . Although _________ contain the same types of molecules as cells, their inability to reproduce themselves by their own efforts means that they are not considered living matter.
micrometers; millimeter; amino acids; DNA; viruses
Prion
misfolded proteins that can cause diseases and form aggregates that damage cells and even whole tissues
How do eukaryotes divide?
mitosis and meiosis
Chaperone proteins
molecule that steers proteins along productive folding pathways, helping them to fold correctly and preventing them from forming aggregates inside the cell. They play a role in the ER also by holding misfolded proteins in the lumen instead of allowing them to be transported in vesicles to the Golgi
Generate movement in cells and tissues
motor proteins
If we say that "x is a ____________ condition for y," we mean that if we don't have x, then we won't have y. Or put differently, without x, you won't have y, but x does not guarantee y.
necessary
Proteins moving from the cytosol into the nucleus enter through a ___________ whereas proteins moving from the cytosol in the ER, mitochondria, or chloroplast enter through __________ located in the membrane.
nuclear pore; protein translocators
The backbone model of proteins shows what?
overall organization of the protein making it easy to compare that protein of interest to other proteins
A molecule moves down its concentration gradient by __________ (passive/active) transport, but requires _____________ (passive/active) transport to move up its concentration gradient. Transporter proteins and ion channels function in membrane transport by providing a _______________ (hydrophobic/hydrophilic) pathway through the membrane for specific polar solutes or inorganic ions. __________ (ion channels, transporters) are highly selective in the solutes they transport, binding the solute at a specific site and changing conformation so as to transport the solute across the membrane. On the other hand, _____________ (ion channels, transporters) discriminate between solutes mainly on the basis of size and electrical charge.
passive; active; hydrophilic; transporters; ion channels
Cells that have been homogenized and centrifuged will be found in the________ (supernatant/pellet) of the test tube.
pellet
Chloroplasts
perform photosynthesis in plant cells
What is homogenization?
preparation of a suspension of cell constituents from tissue by physical treatment in a liquid.
A protein's amino acid sequence is known as its______________ structure.
primary
In affinity chromatography, what binds to the column?
proteins that can attach to the molecule that is attached to the column matrix
Fibrous proteins
proteins that have a simple, elongated 3D structure that have roles in the cell that require them to span a large distance
Detect signals and transmit them to the cell's response machinery
receptor proteins
On removal of urea, an unfolded protein can become _________. The final folded conformation adopted by a protein is that of ________ energy.
renatured; lowest
Differential centrifugation
repeated centrifugation at progressively high speeds to fractionate cell homogenates into their components.
The α helices and β sheets are examples of protein________ structure.
secondary
Proteins are transported out of a cell via the_________ or __________ pathway. Fluids and macromolecules are transported into the cell via the __________ pathway. All proteins being transported out of the cell pass through the ______________ and the __________. Transport vesicles link organelles of the _____________ system. The formation of __________ stabilizes protein structure.
secretory; exocytic; endocytic; ER; golgi; endomembrane; disulfide bonds
The wire model of proteins shows what?
shows position of amino acid side chains. It is good for making predictions of amino acids and how they are involved in the protein's activity
The space filling model of proteins shows what?
shows surface which amino acids are on the surface of the protein
Carry extracellular signals from cell to cell
signal proteins
Proteins move throughout the cell and ultimately arrive at their destination under the direction of _____________. These consist of amino acids and may or may not be cleaved from the protein once it has arrived at its target destination.
signal sequences (without a signal sequence, the protein stays in the cytosol)
Endoplasmic reticulum
site where proteins not destined for the cytoplasm and synthesized and site where lipids are made
A newly synthesized protein generally folds up into a _________ conformation.
stable
Store amino acids or ions
storage proteins
Intermediate filaments
strengthen the cell mechanically
Provides mechanical support to cells and tissues
structural proteins
CAP is a transcriptional activator that exists as a homodimer in solution. Each (subunit/domain) of CAP is composed of a ligand binding (subunit/domain) and a DNA-binding (subunit/domain).
subunit; domain; domain (domains are usually responsible for a particular function. A subunit is a single polypeptide chain)
A protein such as hemoglobin, which is composed of more than one protein _____________ , has ______________ structure.
subunit; quaternary
If we say that "x is a ___________ condition for y," then we mean that if we have x, we know that y must follow. In other words, x guarantees y.
sufficient
3 ways that an active transport pump can harvest energy
sunlight, ATP hydrolysis, or a transmembrane ion gradient (coupled pumps)
Gain-of-function mutations
tend to be dominant because one copy of the mutant allele is sufficient for a mutant phenotype.
Loss-of-function mutations
tend to be recessive because one copy of the mutant allele is not sufficient for a mutant phenotype.
The three-dimensional conformation of a protein is its _________ structure.
tertiary
In a florescent microscope, what is the component that promotes an interaction between the sample and the energy?
the beam splitting mirror
In a light microscope, what is the component that promotes an interaction between the sample and the energy?
the condenser
At any given time, the membrane potential depends on what?
the distribution of ions and the permeability to ions.
What is the difference between an microscope that uses electrons as energy compared to one that uses light for energy?
the electron microscopes allow us to see more detail
Proteins can fold in numerous ways but shape is constrained by what?
the interactions that form within the protein.
What provides magnification in all microscopes?
the objective lens
Globular proteins
the polypeptide chain folds up into a compact shape with an irregular surface
What is the detector in microscopes?
the screen and the eyepiece
Sonication
the use of high frequency sound waves to split open a cell
Denaturation
to cause a dramatic change in the structure of a macromolecule by exposing it to extreme conditions that usually results in loss of biological function; caused by treatment with a solvent that disrupts the noncovalent interactions holding the folded chain together
Why does a red blood cell plasma membrane need transmembrane proteins?
transmembrane proteins anchor the membrane to the underlying cell cortex to strengthen the membrane to withstand the forces and to transport ions and nutrients across the membrane
t-SNAREs
transmembrane proteins on the target membrane that interact with complementary v-SNAREs on the vesicle to provide additional recognition after the tethering protein has captured a vesicle by grabbing hold of its Rab protein and firmly dock the vesicle in place and catalyze the membrane fusion required for the transport vescicle to deliver its cargo
v-SNAREs
transmembrane proteins on the vesicle that interact with complementary t-SNAREs on the target membrane to provide additional recognition after the tethering protein has captured a vesicle by grabbing hold of its Rab protein and firmly dock the vesicle in place and catalyze the membrane fusion required for the transport vescicle to deliver its cargo. They are integrated into the membrane of there ER during their synthesis and are then carried by transport vesicles to their destination
What are the 4 ways that membrane proteins can associate with the lipid bilayer?
transmembrane, monolayer associated alpha helix, lipid linked and protein attached
There are several ways that membrane proteins can associate with the cell membrane. Membrane proteins that extend through the lipid bilayer are called __________ proteins and have__________ regions that are exposed to the interior of the bilayer.
transmembrane; hydrophobic
Carry small molecules and ions
transport proteins
Peroxisomes
use O2 to oxidize dangerous substances
What is a light-driven pump?
uses energy derived from sunlight to drive uphill transport; found mainly in bacteria
What are ways that channels can be opened or closed by the cell?
voltage, ligand, mechanical
A widely used analytical technique used to detect proteins in a sample of homogenate or cell extract
western blotting
What is simple diffusion?
when molecules can cross the membrane without use of a protein to transport them and they go down their concentration gradient