Cell Bio Chapter #4 Review

The breaking open or lysing of the cells to release their contents to produce a cell homogenate or extract

What is the first step in protein purification?

Enzymes

These are proteins whose functions need to be started with ligand binding. These are responsible for nearly all of the chemical transformations that occur in cells. These act as catalysts that permit cells to make/break covalent bonds. These use mechanisms to lower activation energy and speed up the reactions they catalyze

Disulfide bonds

These are the most common covalent cross-links in proteins. These bonds are formed before a protein is secreted by an enzyme in the endoplasmic reticulum that links together two -SH groups from cysteine side chains that are adjacent in the folded protein

Receptor tyrosine kinases

These are transmembrane proteins that are stimulated by extracellular signals which cause receptor proteins to phosphorylate the molecules on certain tyrosines. The phosphorylated tyrosines are then docking sites that bind and activate other proteins

Covalent bonds

These bonds hold peptides together. Heat dissociates noncovalent bonds and attractions, but it does not dissociate these bonds

Noncovalent interactions - Weaker than covalent bonds, so it takes many of them to hold proteins together - Stability of folded shape determined by combined strength of large numbers of noncovalent bonds - Ability of a protein to bind selectively and with high affinity to a ligand is due to the formation of a set of weak, noncovalent interactions

These bonds that help proteins fold and maintain shape include H bonds, electrostatic attractions, van der Waals attractions, and hydrophobic forces

Small molecules

These can be bound to proteins to add extra functions. Examples of these include heme groups in hemoglobin, retinal in the photoreceptor rhodopsin, and coenzymes or cofactors for enzymes

Peptide bonds

These covalent bonds hold protein molecules made of long chains of amino acids together

Van der Waals attractions

These noncovalent interactions are common between molecules that are close together

Electrostatic attractions

These noncovalent interactions are dipole-dipole interactions for charged atoms

Biological properties

These properties of a protein molecule depend on its physical interaction with otehr molecules

Chaperone proteins - Final 3D conformation of proteins is specified by amino acid sequence, but chaperones make folding process more efficient and reliable

These proteins assist in protein folding in living cells. There are two types of these: Some bind to partly folded chains and helps them to fold along the most energetically favorable pathway; others form isolation chambers in which single polypeptide chains can fold without the risk of forming aggregates in the crowded conditions of the cytoplasm

Unstructured proteins

These proteins do not give secondary, tertiary, or quaternary structures; these are only primary amino acid structures that are not bound to anything

Motor proteins

These proteins generate the forces responsible for muscle contraction and most other eukaryotic cell movements. These power intracellular movements of organelles and macromolecules, help chromosomes move to opposite sides of the cell in mitosis, and move organelles along cytoskeleton tracks

Coenzymes

These small organic molecules are used by enzymes which do similar things. Biotin is found in enzymes that transfer a carboxyl group from one molecule to another

Heme groups

A molecule of hemoglobin carries four of these noncovalently bound small molecules which are ring-shaped, each having a single central iron atom. These give hemoglobin and blood their red color, and by binding reversibly to dissolved oxygen through tis iron atom, this enables hemoglobin to pick up oxygen in the lungs and release it to tissues that need it

H bonds

Alpha helices and beta sheets are common folding patterns because they result from the ____________ that form between N-H and C=O groups in the polypeptide backbone

Ligand

Any substance that is bound by a protein as a __________ for that protein

Regulatory site

Common types of control occur when a molecule other than a substrate specifically binds to an enzyme at a special __________ ___________, altering the rate at which the enzyme converts its substrate to a product

1) Newly synthesized, partially folded protein enters the chaperone protein 2) One polypeptide chain is sequestered by the chaperone 3) Chamber cap comes and isolates the chain so it folds correctly 4) Correctly folded protein is released when the cap dissociates - This takes longer and uses more energy, but it is good for proper folding with less errors

Describe the method of chaperone proteins where protein chains are isolated

Chaperone proteins directly bind to newly synthesized polypeptide chains. This is a faster process and takes less energy, but more errors are likely to occur

Describe the method of chaperone proteins which work directly on polypeptides

Substrates

Enzymes bind to one or more ligands, called ___________, and convert them into chemically modified products and doing this continuously without themselves being changed.

1. A cell homogenate is centrifuged at a low speed. This produces the first supernatant and the first pellet. The first pellet contains whole cells, nuclei, and cytoskeletons 2. Supernatant #1 is centrifuged at a medium speed to produce supernatant #2 and pellet #2. Pellet #2 contains mitochondria, lysosomes, and peroxisomes. 3. Supernatant #2 is centrifuged at a high speed to produce supernatant #3 and pellet #3. Pellet #3 contains closed fragments of the endoplasmic reticulum and other small vesicles 4. Supernatant #3 is centrifuged at very high speeds to produce pellet #4. Pellet #4 contains ribosomes, viruses, and large macromolecules

Explain the process of differential centrifugation

Negative regulation

Feedback inhibition is a form of __________ __________: it prevents an enzyme from acting

Transition state Active site

For a colliding water molecule to break a bond linking two sugars, the polysaccharide molecule has to be distorted into the ___________ ___________ in which the atoms around the bond have an altered geometry and electron distribution. This requires a large input of energy, so the enzyme lysozyme is used. Lysozyme has a binding site on its surface, the ____________ ___________, which is where catalysis occurs

Subunit

If a binding site recognizes the surface of a second protein, the tight binding of the second folded polypeptide chain at this site will create a larger protein, whose quaternary structure has a defined geometry. Each polypeptide chain in such a protein is a ___________ and each one may have more than one domain

Antibodies

Immunoglobulin proteins produced by the immune system in response to foreign molecules, especially those on the surface of invading microorganisms. Each one of these binds to a particular target molecule extremely tightly, either inactivating the target directly or marking it for destruction

Conformational change

In allostery, the interactions between sites located in different regions on protein molecules depend on a __________ change in the protein. Binding of a ligand to one of the sites causes a shift in the protein's structure from one folded shape to a slightly different folded shape, altering the shape of the second binding site that can be far away

Amyloid structures

Incorrectly folded proteins can form ____________ structures that damage cells and tissues which contributes to neurodegenerative disorders like Alzheimer's or Huntington's diseases

SDS-PAGE

Individual polypeptide chains from a complex with negatively charged molecules of SDS and migrate as negatively charged SDS-protein complexes through a slab of porous polyacrylamide gel

Prions - Misfolded prion forms of proteins can convert properly folded versions of the proteins inside an infected brain into the abnormal conformation. This causes mislfolded proteins to form aggregates which spread from cell to cell - These can be passed from an affected individual to a normal individual

Infections neurodegenerative diseases like bovine spongiform encephalopathy and Creutzfeldt-Jakob disease in humans are caused by these misfolded proteins

Allosteric

Most protein molecules are __________: they can adopt two or more slightly different conformations, and their activity can be regulated by a shift from one to another

Protein misfolding

Normal proteins can sometimes adopt an abnormal, misfolded prion form. An abnormal prion form of protein turns normal proteins it comes in contact with into prions. This is an example of what? This is one of the causes of Alzheimers, Parkinson's, polyglutamine diseases, amyotrophic lateral sclerosis, and prion diseases

Globular proteins

Protein where the polypeptide chain folds up into a compact shape like a ball with an irregular surface. Enzymes tend to be these types of proteins that are quaternary structures with rounded ball shapes

Chromatography

Proteins purified by a series of __________ steps which use different materials to separate the individual components of a complex mixture into fractions based on the properties of the protein

Fibrous proteins

Proteins which have relatively simple, elongated 3D structures

Positive allostery

The binding of a regulator activates the enzyme by changing the shape of active sites

Allosteric enzyme control - Enzymes can be present in a system, and allosteric regulators make it more selective so it does not need to synthesize new enzymes - Enzymes have at least two different binding sites: active site that recognizes the substrates and one or more sites that recognize regulatory molecules

The binding of another molecule to somewhere that is not the active site changes the shape of the active site so the ligand cannot bind or a different molecule can bind. The small molecule binds to the allosteric site. This can be positively or negatively regulated

Ubiquitin

The covalent modification of attachment of this molecule can target a protein for degredation

Negative allostery

The enzyme is active in absence of a regulator, but when the regulator binds, it becomes inactive as the active site shape changes

Positive regulation

The enzyme's activity is stimulated by a regulatory molecule rather than being suppressed. This occurs when a product in one branch of the metabolic maze stimulates the activity of an enzyme in another pathway

Free energy (ΔG)

The final folded structure, or conformation, of the protein is in a shape in which the ___________ ___________ is minimized. Folding proteins is energetically favorable and releases heat as well as increases entropy of the universe

Folding pattern of a protein

The predominant way to discover the __________ _________ is by using x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy (cryo-EM)

Beta sheet

These are made when H bonds form between segments of a polypeptide chain that lie side-to-side. Come in parallel and antiparallel types, but both produce rigid, pleated structures that form the core of many proteins

Side chains

These are parts of amino acids that project from the backbone and are not involved in forming peptide bonds. These give amino acids unique properties, e.g. nonpolar/hydrophobic, negatively or positively charged, or uncharged polar

GTP-binding proteins

This acts as a molecular switch; they are in their active conformation when GTP is bound, but they can hydrolyze their GTP to GDP which releases a phosphate and flips the protein to an inactive conformation. This method of protein control uses a phosphate group that is not enzymatically transferred from ATP to the protein. Instead, the phosphate is part of a guanosine nucleotide (GTP) that binds tightly to this protein. The process performed by this protein is reversible as the active conformation is regained by dissociation of GDP and the binding of new GTP

ATP hydrolysis

This allows motor proteins to produce directed movement in cells. Normally, a protein that walks along cytoskeletal fibers moves through conformational changes but nothing drives the changes in one direction or another, so it is reversible. The movement through conformational changes must be unidirectional to create irreversibility, so the movement is done by coupling one of the conformational changes to this process. Through this, a lot of free energy is released, making it unlikely that the protein will undergo a reverse shape change that would be needed to move backwards

Palmitate

This covalent modification is the addition of the fatty acid ___________ to a cysteine side chain which drives a protein to associate with the cell membrane

p53

This covalent modification occurs when many proteins are modified on more than one amino acid side chain. This occurs with ___________ protein which helps control how a cell responds to DNA damage and other stressors can be covalently modified of 20 sites

Specificity

This describes how each protein molecule can bind to just one or a few molecules out of the many thousand different molecules it encounters. Binding of a protein to other molecules shows great __________

SDS

This detergent is used in polyacrylamide gel electrophoresis and solubilizes proteins for the process

Lysozyme - In the absence of lysozyme, energy of random molecular collisions almost never exceeds the activation energy required for the reaction to occur. Hydrolysis of the polysaccharide would occur extremely slowly if at all - For lysozyme, the active site can also contain precisely positioned chemical groups that speed up the reaction by altering distributions of electrons in the substrates - Binding to the enzyme changes the shape of the substrate and drives the bound molecule toward a certain transition state

This enzyme severs the polysaccharide chains that form the cell walls of bacteria - bacterial cell walls are under pressure due to intracellular osmotic forces, so cutting small numbers of polysaccharide chains causes the cell wall to rupture and the bacterium to lyse. This is an enzyme that acts as a natural antibiotic in egg whites, saliva, tears, and other secretions. This was the first enzyme to have its structure on its atomic level marked out by X-ray crystallography. This greatly reduces the activation energy needed for hydrolysis to take place so that the overall chemical reaction occurs millions of times faster in the presence of this enzyme than if it was in its absence

Secondary structure

This includes alpha helices and beta sheets that form within certain segments of the polypeptide chain; these folds are part of the ___________ structure

Electrophoresis - If too many proteins in the sample or if they are similar in migration rates, 2D gel electrophoresis is used to resolve them

This is a method of separating proteins where a mixture of proteins is loaded onto a polymer gel and subjected to an electric field; the polypeptides will migrate through the gel at different speeds depending on their size and net charge. This produces bands visible by staining, and each band is for a different protein. This is used when an electrical field is applied to a solution of protein molecules and the proteins migrate in a direction and at a speed that reflects their size and net charge.

Mercaptoethanol

This is a reducing agent that is used to break disulfide bonds within or between proteins. This allows unfolded polypeptide chains to migrate at a rate that reflects their molecular weight with the smallest proteins migrating most quickly. This is a treatment so that SDS-PAGE can be completed

Hydrophobic force

This is a weak interaction which has a central role in determining the shape of proteins. This causes nonpolar/hydrophobic side chains to cluster in the interior of a folded protein and polar side chains to arrange at the outside of the folded protein where they can H bond with water and other polar molecules

Carboxypeptidase

This is an enzyme that cuts polypeptide chains and carries a zinc ion in its active site. During cleavage of the peptide bond by this enzyme, the zinc ion forms a transient bond with one of the substrate atoms, assisting the hydrolysis reaction

Protein domain

This is an organizational unit of a protein defined as any segment of a polypeptide chain that can fold independently into a compact, stable structure

Binding site

This is any region on a protein's surface that interacts with another molecule through sets of noncovalent bonding. Region of a protein that associates with a ligand that usually consists of a cavity in the protein surface formed by a particular arrangement of amino acid side chains

Polypeptide backbone

This is formed by a repeating sequence of the core atoms (-N-C-C-) found in every amino a cid

Antiparallel beta sheet

This is formed when neighboring beta sheets run in opposite directions

Parallel beta sheet

This is formed when neighboring beta sheets run in the same direction

Hydrogen bonding

This noncovalent interaction is when a hydrogen atom that is bonded to a highly electronegative atom is attracted to an unshared pair of electrons of an electronegative atom in a nearby molecule

Homogenization

This is generally the mechanical procedure where the plasma membranes of cells can be ruptured so that the cell contents are released. This includes the breaking apart of cells with high-frequency sounds, mild detergents that make holes in the plasma membrane, forcing cells through small holes using high pressure, and shearing cells between a rotating plunger and the walls of glass vessels. This produces a homogenate/extract with large and small molecules from the cytosol as well as all of the membrane-enclosed organelles

Quaternary structure

This is made of interacting polypeptides that compose a protein molecule complex with more than one polypeptide chain. Different proteins (domains) come and interact with each other

Alpha helix - These have the nonpolar/hydrophobic amino acids facing the inside of the protein

This is made when a single polypeptide chain turns around itself to form a rigid cylinder. An H bond is made between every fourth amino acid, liking the C=O of one peptide bond to the N-H of another. This pattern produces a right-handed ___________ with a complete turn every 3.6 amino acids. It is generated by placing many similar subunits next to one another in the same repeated relationship to the one before. The subunits randomly join in straight lines, so a spiral structure forms.

C-terminus (carboxyl terminus)

This is the end of an amino acid that carries a free carboxyl group

N-terminus (amino terminus)

This is the end of an amino acid that carries the amino group

Tertiary structure

This is the full, 3D conformation formed by the entire polypeptide chain. This is the combination of secondary structures

Affinity chromatography - Large amounts of antibodies that can recognize a protein/antigen can be attached to a matrix of a chromatography column and then be used to extract the protein from a mixture - Can be used to isolate proteins that interact physically with a protein being studies. The purified protein is attached tightly to the column matrix and the proteins that bind to it will remain in the column and can be removed by adding salts or changing pH - Separation based on shape

This is the most efficient form of protein chromatography that separates polypeptides on the basis of their ability to bind to a particular molecule. The column used here contains a matrix covalently coupled to a molecule that interacts specifically with the protein of interest (e.g. antibody or enzyme substrate). The proteins that bind specifically to such a column can be released by a pH change or by concentrated salt solutions

Centrifugation

This is the most widely used procedure to separate a homogenate into different parts/fractions. This can be used with a fixed angle rotor or a swinging-arm rotor. This takes homogenate and produces a supernatant and a pellet

Supernatant

This is the product of centrifugation that contains smaller and less dense components

Primary structure

This is the protein structure's amino acid sequence

Differential centrifugation - Seperation in different densities with different centrifuges and speeds in different tubes

This is the repeated centrifugation at progressively higher speeds which fractionates cell homogenates into their components based on size and density. Larger and denser components experience the greatest centrifugal force and move most rapidly

Antigen

This is the target molecule of an antibody. Antibodies bind to this with specificity

Mass spectrometry - Peptides derived from digestion with trypsin are blasted with a laser. This heats the peptides, causing them to become ionized and ejected in the form of a gas. The electric field accelerates the peptide ions which fly toward the detector. The time it takes to arrive at the detector is related to their mass and charge - The larger the peptide, the more slowly it moves; more highly charged, the faster it moves

This is the technique that determines the exact mass of every peptide fragment in a purified protein, which then allows the. protein to be identified from a database with a list of every protein thought to be encoded by the genome of the relevant organism

Denatured

This is the unfolding of proteins due to treatment with solvents that disrupt noncovalent interactions holding the folded chain together

Protein phosphorylation - The reverse reaction of removal of a phosphate group (dephosphorylation) is catalyzed by a protein phosphatase

This is when the enzyme protein kinase catalyzes the transfer of the terminal phosphate group of high energy ATP to the OH group of a serine, threonine, or tyrosine side chains of the protein

Phosphorylation - Removal of phosphate by a second enzyme restores the protein to original conformation and restores initial activity - Reversible protein phosphorylation controls activity of many proteins in eukaryotic cells

This method can control protein activity by causing a conformational change from attaching a phosphate group covalently to one or more of the protein's amino acid side chains. Each phosphate group has 2 negative charges, and the enzyme catalyzed addition of the group causes a conformational change because now it can attract positively charged amino acid side chains from somewhere in the same protein. The structural shift affects binding of ligands elsewhere on the surface of the protein, altering its activity

Column chromatography

This method of fractioning proteins is used when a mixture of proteins in solution is applied to the top of a cylindrical column filled with a permeable solid matrix immersed in solvent. A solvent is then pumped through the column, and because different proteins are retarded to different extents by their interactions with the matrix, they can be collected separately as they flow out from the bottom. The choice of matrix allows proteins to be separated according to charge, hydrophobicity, size, or affinityThis involves a stationary and a mobile phase.

ATP walk - Muscle protein myosin walks along actin filaments rapidly during muscle contraction

This method of protein control is present in the actin-myosin heads where ATP molecules bind to myosin heads so that they detach from they actin filaments. ATP then dissociates to ADP and phosphate. This process is allowed to happen because calcium releases the troponin/tropomyosin sites, the phosphate leaves, and myosin and actin bind

Feedback inhibition - Works almost instantaneously and is rapidly reversed when product levels fall

This occurs when an enzyme acting early in a reaction pathway is inhibited by a molecule produced later in that pathway. Whenever large quantities of the final product begin to accumulate, the product binds to an earlier enzyme and slows down its catalytic action, limiting further entry of substrates into that reaction pathway. The product of the reaction can limit the formation of other products, i.e. the product inhibits the first step

Covalent modification

This process allows the cell to. optimally se the proteins it produces, and it allows the cell to respond rapidly to changes in its environment

Pellet

This product of centrifugation contains the larger and more dense components

Hydrolysis - Pure polysaccharide can sit in water without being hydrolyzed because there is an energy barrier to the reaction called activation energy

This reaction is catalyzed by lysozyme which adds a molecule of water to a single bond between two adjacent sugar groups in the polysaccharide chain, causing the bond to break. This reaction is energetically favorable because the free energy of the severed polysaccharide chain is lower than the free energy of the intact chain

Swinging-arm rotor

This rotor used in ultracentrifugation has metal buckets that hold the tubes containing the homogenate which are free to swing outward as the rotor turns. This carries smaller volumes but forms a more even pellet.

Retinal

This small molecule allows rhodopsin to detect light, and it is attached to the protein by a covalent bond to a lysine side chain. This changes shape when it absorbs a photon of light, and the change is amplified by rhodopsin to trigger reactions that form electrical signals

Amino acid sequence - Position of each amino acid determines its 3D structure which is stabilized by noncovalent interactions between different parts of the molecules

This specifies the shape of a protein. Polar and nonpolar regions of these determine the folding and structure of proteins in an aqueous environment such as the cytosol. This is the unique order of amino acids in proteins that are the same from one molecule of the protein to the next

Backbone to backbone

This type of H bond in protein that helps stabilize the folded shape is between atoms of two peptide bonds

Backbone to side chain

This type of H bond in proteins that helps stabilize the folded shape is between the atoms of a peptide bond and an amino acid side chain

Sidechain to sidechain

This type of H bond in proteins that helps stabilize the folded shape is between the atoms of two amino acid side chains

Gel-filtration chromatography

This type of chromatography separates proteins based on their size. The matrix consists of tiny porous beads, and the proteins that are small enough to enter the holes in the beads are delayed and travel more slowly through the column. Proteins that cannot enter the beads are washed out of the column first. This allows estimation of protein size. This is also known as size exclusion chromatography

Ion exchange chromatography

This type of chromatography uses columns that are packed with small beads carrying either positive or negative charges that retard proteins of the opposite charge. The association between the protein and the matrix depends on the pH and ionic strength of the solution passing down the column. The positive or negative media holds back the complementary charged protein components. To release the bands, pH is changed to release the bands and amino acids that are held back. This uses separation based on charge

Acetylation

This type of covalent modification is the addition of an acetyl group to a lysine side chain

Fixed angle rotor

This type of rotor used in ultracentrifugation holds larger volumes, but the pellet forms less evenly compared to a swinging arm rotor

Velocity sedimentation (density-gradient centrifugation)

This uses high concentrations of sucrose or cesium chloride gradients and centrifuges them along with sub-cellular components so that they move up or down in the tube to wherever they match the density of the gradient. Bands at the bottom of this have the higher densities. The components are separated by their densities all in one tube

It needs to be refrigerated/cooled because it rotates at a high speed which would produce heat. It also needs to be in a vacuum so friction does not heat up the homogenate

What are the two requirements of an ultracentrifuge's mechanism?

Kinase

What enzyme catalyzes phosphorylation?

Phosphatase

What enzyme catalyzes the removal of a phosphate group, or dephosphorylation?

Vast numbers of polypeptide chains could be made from the 20 amino acids, but only a small fraction are produced by cells because most biological functions depend on proteins with stable, well-defined 3D conformations. Hydrophobicity/hydrophilicity, functions, pH, chaperone proteins, and mutations all affect protein stability and likelihood of existence. Functional proteins cannot engage in unwanted interactions with other proteins in the cell

Why do cells only have a few functional proteins considering that several polypeptides can be formed with amino acids?