BIOL 3090 Exam 1 Study Guide

Why do you use immersion oil with the high power lenses?

Refraction occurs: glass → air → glass Reduce refraction: glass → oil → glass

You should be able to recognize the Michaelis-Menton equation and have an idea of what it's describing.

This equation describes a rectangular hyperbola in terms of the amount of substrate bound to the enzyme (ie the extent of enzyme saturation)

TIRF microscopy to the rescue

Total internal reflection fluorescence (TIRF) microscopy 1. For very narrow focal planes: ex: portions of cells just under the coverslip (very specialized microscopy) 2. Angle of incoming excitation light allows most of it to reflect off the coverslip and back up through the lens 3. However, a narrow band of evanescent (quickly fading) light reaches down only 50-100 nm into the tissue. Only this zone fluoresces red. Everything below it remains dark. 4. Great for the cytoskeleton

Intracellular protein degradation a) Lysosomes

hydrolytic enzymes (hydrolases) - low pH environment (pH = ~5)

Of the principal macromolecules, which has the most diversity in terms of structure and function?

proteins

Know the 20 standard amino acids and their three letter abbreviated names.

****ING DO IT

9) How do proteins fold properly? (Fig. 3-16).

1)Several folding steps occur to get to the native state. 2) Each successive step is thermodynamically more favorable, thus limiting the number of theoretical folds. 3) Notice that secondary structures form before tertiary structures.

What is the activation energy? What does a catalyst do for a reaction that has a favorable -ΔG? What does a catalyst not do?

the minimum quantity of energy which the reacting species must possess in order to undergo a specified reaction. Catalysts lower activation energy.Does NOT alter the change in free energy between reactants and products. Does NOT affect the equilibrium constant, Keq.

Know your milli, micro, nano, and pico Molar scales. At what Kdrange do most biochemical interactions occur?

the nM range (10-9, 10-10)

Which amino acid forms disulfide bonds? Why are Pro and Gly "special"?

Cys forms disulfide bonds. Gly and Pro disrupt alpha-helix formation

What macromolecule would you say indirectly regulates the synthesis of all other macromolecules?

DNA

What's an ES cell, what features does it possess in terms of mammalian development, and why is it in the news? How do you obtain ES cells?

ES cell (embryonic stem cell) - stem cells derived from the inner cell mass of an embryo Totipotent; ethical debates regarding the use of human ES cells Obtain from cells that have been fertilized in vitro iPSCs can reverse adult, mature cells to stem cells - shutdown ethical debate

Proteomics (aka, "biochemistry" in-the-day) -Edman degradation -Time-of-flight mass spec. MOLDI-TOF (Fig. 3-43) -Electrospray ionization (ES) spectrometry (Fig. 3-44)

Edman degradation: Sequential identification of the amino terminal residue which is cleaved and identified by high pressure liquid chromatography. Time-of-flight mass spec. MOLDI-TOF (Fig. 3-43) pulses of light from a laser ionize a protein or peptide mixture that is absorbed on a metal target (step 1). An electric field in the mass analyzer accelerates the ions in the sample toward the detector (steps 2 and 3). The time it takes an ion to reach the detector is proportional to the square root of the mass-to-charge (m/z) ratio. Among ions having the same charge, the smaller ions move faster (shorter time to the detector). The molecular weight of each ion from the sample is calculated using the time of flight of a standard. Electrospray ionization (ES) spectrometry (Fig. 3-44) converts proteins and peptides in a solution into highly charged gaseous ions by passing it through a charged needle - which forms the droplets. Evaporation of the solvent allows the ions to enter the mass analyzer that separates ions based on m/z

Halophiles vs. Thermophiles vs. Methanophiles (Archaea - Extremeophiles)

Halophiles - grow in high salt environment Thermophiles - grow in high temperatures Methanophiles - grow in oxygen deficient environments; they reduce CO2 to make CH4

Why is the intracellular pH so important for protein structure and thus function?

Keeps them at the proper charge and shape Proteins will denature at the wrong pH stopping them from doing their job. Also enzymes and reactions occur at very specific pHs optimally

Isoelectric focusing and SDS-PAGE. What the heck does isoelectric mean?

Lets say your protein of interest is rich in acidic amino acids • (Asp, Glu, Phospo-Ser, Phospho-Thr, Phospho-Tyr) • Its overall net charge at pH 7.2 would be negative. • Now, start reducing the pH (adding protons to solution). • Added protons neutralize the negative charges on acidic AA's. • Let's say at pH = 5.0, the net charge on your protein is 0 (neutral) • pH 5.0 is the iso-electric point (pI) of your protein.

Super-resolution microscopy:

-Achieving resolution below 0.2 um (200 nm) with light microscopy -Down to ~10 mn resolution at best ("truthful hyperbole") -Practically speaking, 20-50 nm in x and y axes, bur ~120 nm in z axis -Different ways of accomplishing super-resolution: 1) SIM 2) STED 3) PALM

Light sheet microscopy:

-Illumination objective scans the sample to generate a light sheet -The orthogonal (Detection objective) lens then detects the fluorescence -Both lenses move coordinately in steps -Stacks of images (sheets) are assembled by computer to give a 3D rendering (image) -Zebrafish brain with Ca++ (calmodulin) imaging

Basic definitions of Vmax, Kcat, and Km. The relationship between Km and affinity.

-Km: the Michaelis Constant measures the affinity that an enzyme has for its substrate. -Vmax: measures the maximum velocity of the reaction when the substrate concentration [S] is saturating with respect to what the enzyme can handle. -kcat: rate constant = the number of substrate molecules processed per enzyme molecule per second. a.k.a. the "turnover number" -Lower Km - higher affinity -Higher Km - lower affinity

Bright-Field microscopy

-Standard medical student microscope -Very little detail, unless specimen is fixed, sectioned, and stained -Used in histology, anatomy -Rarely used in molecular-cell research

What non-covalent allosteric modifications did we describe?

1) Ca++ binding to calmodulin, a co-factor for several other functional (target) proteins 2) Guanine nucleotide switch proteins such as Ras, Ran, many others. 3) Proteolytic cleavage: - to activate or inactivate a protein's function

What is a Pulse-Chase experiment?

1) Incubate cells for a short time (say 5 min) with 35S-Met. Proteins synthesized in this 5 min time window are radio-labeled. 2) Wash cells (at 4oC) to get rid of unincorporated 35S-Met. 3) Add back normal Met that is not radio-labeled (37oC) Protein synthesis resumes, but all proteins made now are unlabeled. 4) Observe what happens to the radio-labeled proteins. Are they: post-translationally modified? secreted? localized to an organelle? can not post-translationally modified

Which isotope would you use to label DNA? A protein during its synthesis in the cytoplasm?

1. 35S-methionine and 35S-cysteine for labeling proteins. 2. 125-I for protein (tyrosine) labeling. (Use a chemical Rx to link I to Tyr). 3. 14C or 3H-nucleotides for nucleic acids; 14C or 3H-AAs for proteins. 4. 32P-ATP for phosphorylation of proteins or nucleic acids. (α, β or γ PO4?)

What are the four aspects of the cell theory. (with people)

1. All living things are composed of cells and their products. (Schleiden and Schwann, 1838) 2. Cells are essentially alike in chemical constitution. Consider glycolysis in bacterial cell vs. human cell 3. "Omnis cellula e cellula" (Virchow, 1855) - All cells come from cells 4. The activity of the organism as a whole is the sum of the activities and interactions of essentially independent cell units (activity = development, physiology, etc.

Review the model organisms and what features each organism is good for.

1. Budding yeast: most primitive eukaryote (diploid or haploid) Control of cell cycle and cell division Aging Chromosome structure 2. Alga (Bio-Fuels - ExxonMobil) Photosynthesis 3. Roundworm Cell lineage (trace back every adult cell) Aging 4. Fruit fly Remarkably similar genes to vertebrates Cells express different genes 5. Planarian Regeneration What gene is involved in this regeneration? 6. Zebrafish (vertebrate) 7. Mouse (mammal) Closest thing to humans 8. Plant 9. Viruses

Protein motifs and domains (Fig. 3-10, 3-11, and 3-12).

A protein motif is a region of the protein that contains various secondary structures. Has its own defined 3D structure and topology. Has its own defined function versus other motifs within the same protein. The definition of domain is similar to "motif" bc they both contain secondary structures. Domains are usually larger than motifs and could contain several motifs.

Positive/negative ΔG's and coupled reactions. ATP hydrolysis to drive coupled reactions forward.

A positive ∆G means you have to put energy into a reaction in order for the reaction to go forward: (Endergonic)A negative ∆G means that energy was released from the reaction: (Exergonic)

Know what atoms make up the peptide bond and how these atoms contribute to secondary structures (Fig. 3-3a, b, and c). What are the phi and psi bonds?

Atoms that make up the peptide bond: carboxyl group and amide group (H, C, N, O) The carbonyl O is H bonded to amide N four peptide bonds downstream Phi bond - N to alpha C Psi bond - Alpha C to C

What other proteins or protein complexes help nascent fold proteins, and how do they work (Fig. 3- 17a and 3-18)?

Chaperones: A family of proteins that facilitate the folding of most cellular proteins. Chaperonins: protein complexes that directly facilitate folding.

Confocal, deconvolution

Confocal Microscopy (two types): 1. Laser-scanning confocal microscope -Scanning confocal uses a single point of light that scans in a raster -Single pinhole excludes unwanted fluorescence (relatively slow) 2. Spinning disk confocal microscope -Spinning disk confocal uses multiple points of light to scan the specimen -Multiple pinholes exclude unwanted fluorescence (relatively fast)

What important internal features distinguish eukaryotic cells from prokaryotic cells?

Eukaryotic organelle membranes establish essential environments for specialized functions that must be separated from the cytosol A true nucleus that contains chromosomes, the nucleolus, and other nuclear components

T/F: Right-handed (D) amino acids make up proteins.

False: Only L-amino acids are used for protein synthesis

What is fixation and embedding? What machine do you use to section embedded tissues?

Fixation: formaldehyde and/or glutaraldehyde to cross-link macromolecules, "fixing" components in place. Osmium tetroxide fixes membranes (lipids). Nasty chemical; covalent cross links Embedding: fixed tissue is dehydrated with increasing concentrations of ethanol (30, 50, 75, 95, 100, 100% EtOH) Then infiltrated with liquid waxes or liquid plastic resins that then polymerize using an ultra-microtome. You can make your own glass knife or purchase a diamond knife.

What is FRAP?

Fluorescence Recovery After Photo Bleaching

What inherent problem exists in the conventional fluorescence microscope shown in this figure? (Fig. 4-17)

Fluorescence from multiple focal planes

Reaction rates and chemical equilibrium. Why is the Kd useful in Cell Biology/Biochemistry?

Forward and reverse rates are different prior to equilibrium, but equal to each other at equilibrium. The Kd is a measure of how tightly the protein binds to the DNA. The lower the Kd, the tighter the interaction. The higher the Kd, the weaker the interaction.

What it FRET? What is?

Foörster Resonance Energy Transfer To determine protein-protein interactions in vivo We can do this too with our Leica SP8 confocal

In vitro peptide synthesis: what's unusual about this? X-ray crystallography. (Fig. 3-45) What's a synchrotron?

In vitro peptide synthesis: what's unusual about this? In vitro synthesis is "backwards from the carboxyl end to the amino terminal end. X-ray crystallography. (Fig. 3-45) What's a synchrotron? an extremely powerful source of X-rays. The X-rays are produced by high energy electrons as they circulate around the synchrotron. •Determines the precise three-dimensional structure of a protein (down to the atom).

Know these terms: ligand substrate affinity specificity CDR antigen epitope active site catalytic site

Ligand: molecules that bind to a protein with great specificity. : analogy of a lock and key. Substrate: a reactant molecule that binds to an enzyme. : could be a small molecule or a large macromolecule. Affinity: strength of binding between protein and ligand. : Keq (or KD) is a measure of affinity. Specificity: ability of a protein to bind one molecule vs. many others. CDR = Complementarity Determining Region; aka, antigen binding site Antigen = foreign material that is specifically bound by the CDRs. Epitope = site on the antigen that the antibody specifically binds. Active site of the enzyme consists of a substrate binding site and a catalytic site catalytic site: Catalytic residues of the site interact with the substrate to lower the activation energy

Difference between magnification and resolution. What factors are important for best resolution? Maximum resolution for a good light microscope is _________?

Magnification: Total Mag. = (mag. of objective lens) X (mag. of ocular lens) Resolution: the ability to see two closely spaced objects as separate The most important feature of the microscope is resolution max 0.2

van der Waals and hydrophobic interactions. (Figs. 2-10 and 2-11 are good). - Why are transient bonds important for the cell?

Non-specific interactions between two closely spaced atoms. Occur due to random fluctuations in electron distributions around the atoms involved can be cumulative to help stabilize macromolecular structure. (e.g., the double-stranded DNA helix)

What is the nucleoid in a prokaryotic cell (Fig. 1-11)?

Nucleoid - region of the cell containing DNA Prokaryotes have no true nucleus; they have a nucleoid, but no surrounding membrane

Homologous proteins: what's the difference between orthologous proteins vs. paralogous proteins?

Orthologs are corresponding genes in different lineages and are a result of speciation, whereas paralogs result from a gene duplication.

Primary and secondary structures (Figs. 3-4, 3-5, and 3-6),

Primary structure: linear sequence of amino acids. Secondary structures: alpha (a) helix and beta (b) strands. Primary forms secondary through hydrogen bonds

What are prions, and what does it mean when we say prion are contagious when misfolded?

Prions: misfolded proteins that are infectious.

Review the eukaryotic cell cycle. What's G0? Roughly, what percentage of the cell cycle is mitosis?

Programmed by genes and executed by the proteins (cycle varies by cell type) Liver and brain cells are in G0 phase so they do not divide G0 phase = resting phase; not replicating during this stage Percentage of cell cycle that is mitosis: 10%

What interactions help fold proteins into tertiary structures (Fig. 2.11 and Fig. 3-7) and quaternary structures?

Protein tertiary structure is the three dimensional shape of a protein. Quaternary structure: one protein interacts with another protein by the same set of interactions used for tertiary structure. Both are Formed by Hydrophobic interactions, Hydrogen bonds, Ionic interactions, Disulfide

What are Guanine nucleotide switch proteins?

Ras, ran Considered to be inactive when they have GDP bound

Electrophoresis: Ionic and non-ionic detergents.

SDS is an ionic detergent that denatures the proteins, and coats them, giving them an overall negative charge

Cryoelectron microscopy making a huge come-back (Fig. 3-46).

Somewhat lower-level resolution of protein/particle structure. Tremendous recent improvements. Protein/particle is frozen in liquid helium and examined in its frozen, hydrated state by cryo-EM. Images are captured on an extremely sensitive camera (detector) (mega $$$). Sophisticated computer programs then determine 3D structure from 1000's of imaged particles

two photon

Two photon excitation microscopy (still confocal) 1. The cone of 488 nm in regular point-scanning confocal microscopy may cause photo-bleaching (photo-toxicity) due to its relatively high energy 2. To solve this, use two photons of 960 nm (but half the energy). Focus them on one spot in a plane such that they are in sufficient density and arrive on the fluorescent molecule at the same time (within a femtosecond). 3. Achieve the same excitation and emission, but without the photo-toxicity 4. Allows deep penetration of excitation wave-length light into living tissue

What happens when you: acetylate the side chain of Lysine (Lys); phosphorylate Ser's side chain.

When the (basic) lysine is acetylated and is transformed to acetyl lysine it becomes acidic. The polar serine with an uncharged R group becomes acidic after phosphorylation

Methods for protein purification (continued) Column chromatography (Fig. 3-40a, b, and c): a) gel filtration b) ion-exchange c) affinity chromatography

a) gel filtration: separation based on size - large proteins elute first b) ion-exchange: separation based on charge c) affinity chromatography: separation based on differences in binding interaction with a ligand that is immobilized to a stationary material

Refraction

caused by light moving from one refractive index into a different refractive index Light bends when it moves from one refractive index into another Light moves more slowly through regions of high refractive index, shifting phase

Diffraction

constructive and destructive interference

Henderson-Hasselbalch equation, and what it means regarding biological buffers (pKa). When does the pKa = the pH? What is the middle pKa of phosphoric acid, and why is that one so important?

explains how weak acids work as buffers, relating their pKa's to effective buffering capacities within the pH scale. pKa=pH the ratio [A-]/[HA] = 1, and the log of 1 = 0. Instead, our cells use phosphoric acid as the intra-cellular buffer because it's middle pKa is 7.2.

The ubiquitin pathway: Ubiquitin... E3 ligases... Proteasome... What happens to ubiquitin at the proteasome?

is covalently attached to a target protein destined for destruction link ubiquitin to lysine residues within the target proteins is like a garbage disposal in the cytoplasm. Proteosome is like a garbage disposal in the cytoplasm; the ubiquitin is released alongside the proteins and goes back to start the cycle of marking proteins again

What does SDS do to proteins? Does SDS break covalent bonds?

•SDS disrupts hydrophobic interactions within folded proteins; • SDS does not break covalent bonds Also reference above

Archaea are in between prokaryotes and eukaryotes in terms of evolution (Fig. 1-1).

- Eukaryotes and Archaea diverged from the bacteria before they diverged from each other - Archaea show many similarities to the eukaryotes - A "primitive" cell (referred to by Lodish) is the cell is the closest thing to an autonomous biological unit that exists and is the fundamental unit of life. The last common ancestor of all life on earth was a cell, and at the cellular level all life is remarkably similar.

Post-translational modifications: covalent acetylation, phosphorylation, methylation, glycosylation. - The effect of acetylating Lysine's side chain. - What amino acids can be phosphorylated? - What are kinases? What are phosphatases? - What amino acids can be methylated? What does methylation do for the side chains? - What amino acids are glycosylated (e.g., O-linked versus N-linked)?

- The effect of acetylating Lysine's side chain. Turns the basic lysine to an acidic acetyl lysine - What amino acids can be phosphorylated? on/off switch -- Tyr, Ser, or Thr -- phosphorylating makes acidic - What are kinases? What are phosphatases? Kinases phosphorylate (on) and phosphatases dephosphorylate (off) - What amino acids can be methylated? What does methylation do for the side chains? lysines, arginines, and histidines -- methylating makes hydrophobic - What amino acids are glycosylated (e.g., O-linked versus N-linked)? •O-linked glycosylation on Ser and Thr • N-linked glycosylation on Asparagine (Asn) Increases mass substantially

Definition of pH, both the little mathematical formula for pH, and what the formula means.

pH = concentration of the hydrogen ion pH=-log[H+]

Differences in Electronegativity to explain: polar versus non-polar covalent bonds ionic bonds hydrogen bonds

polar versus non-polar covalent bonds Covalent bonds: sharing of electrons to fill orbitals Nonpolar is when the differences in electronegativity is identical or very close polar is when the differences in electronegativity is significantly different ionic bonds: electrons of one atom are transferred permanently to another atom. hydrogen bonds: a weak bond between two molecules resulting from an electrostatic attraction between a proton in one molecule and an electronegative atom in the other.

The difference between true equilibrium and steady state.

true equilibrium is a dynamic state in which reactants are being converted into products at all times, but at the exact rate that products are being converted back into reactants. BUT intermediate products - AB and ABC - never reach a true equilibrium because they're rapidly consumed. But, products AB and ABC reach what is called a STEADY STATE Where the [AB] and [ABC] remain constant.

Extracellular proteases and peptidases Intestines contain trypsin and chymotrypsin:

• endoproteases that cleave within the protein next to basic or hydrophobic AA's. • exopeptidases cleave from the ends of the protein. Peptidases cleave small peptides down to tri/dipeptides then to individual AAs

Tandem MS/MS and then Liquid Chromatography MS/MS (Fig. 3-47) NMR spectroscopy

•Uses strong magnetic fields to change the spin on atoms. • Effect of this change is detected in neighboring atoms. • Distance between atoms is determined. • Structures determined from collected data.

Centrifugation: Differential versus rate-zonal versus equilibrium density (Figs. 3-37 and 4-37). What are Svedberg units?

•sedimentation velocity ÷ centrifugal force

List the names and functions of the various eukaryotic organelles (Fig. 1-12b). Would you consider the nucleolus inside the nucleus an organelle? Golgi complex secretory vesicles peroxisomes cytoskeletal fibers microvilli cell wall vacuole chloroplasts plasmodesmata Nucleolus

1. Golgi complex: processes and sorts secreted proteins, lysosomal proteins, and membrane proteins synthesized on the rough ER 2. Secretory vesicles: store secreted proteins and fuse with the plasma membrane to release their contents 3. Peroxisomes: contain enzymes that break down fatty acids into smaller molecules used for biosynthesis and also detoxify certain molecules 4. Cytoskeletal fibers: form networks and bundles that support cellular membranes, help organize organelles, and participate in cell movement 5. Microvilli: increase surface area for absorption of nutrients from surrounding medium 6. Cell wall: helps maintain the cell's shape and provides protection against mechanical stress 7. Vacuole: stores water, ions, and nutrients, degrades macromolecules, and functions in cell elongation during growth 8. Chloroplasts: surrounded by a double membrane and contain a network of internal membrane-bounded sacs (carry out photosynthesis) 9. Plasmodesmata: tubelike cell junctions that span the cell wall and connect the cytoplasm of adjacent plant cells 10. nucleolus: NOT an organelle; not bound by a membrane

What is the Green Fluorescent Protein and its derivatives, and how can we use them? (Fig. 4-16)

1. Green Fluorescent Protein (GFP): a naturally-occuring fluorescent protein in the jellyfish, Aequorea victoria 2. Use GFP as a tag for your favorite cellular protein Refer to it now as a fusion (or chimeric) protein 3. How do you make a GFP fusion protein? Ligate the cDNA that encodes GFP to the cDNA that encodes your protein; maintain open reading frames: then express the chimeric protein from the ligated cDNA that is introduced into cells 4. Allows you to locate your protein using fluorescence microscopy

What are the three components of the cytoskeleton (Fig. 1-13), and what proteins make up these components?

1. Microtubules: made of alpha and beta tubulin 2. Intermediate filaments: IF proteins, many different kinds 3. Microfilaments: made of actin only

List the names and functions of the various eukaryotic organelles (Fig. 1-12b). Would you consider the nucleolus inside the nucleus an organelle? Plasma membrane mitochondria lysosome nuclear envelope nucleolus nucleus smooth ER rough ER

1. Plasma membrane: controls movement of molecules in and out of the cell and functions in cell-cell signaling and cell adhesion. 2. Mitochondria: generate ATP by oxidation of glucose and fatty acids (surrounded by a double membrane) 3. Lysosomes: degrade material internalized by the cell and worn-out cellular membranes and organelles (have an acidic lumen) 4. Nuclear envelope: encloses the contents of the nucleus (double membrane); the outer nuclear membrane is continuous with the rough ER 5. Nucleolus: nuclear subcompartment where most of the cell's rRNA is synthesized; not a typical organelle because it has no lipid membrane, making it one of the few non-membrane bound organelles in the cell 6. Nucleus: is filled with chromatin composed of DNA and proteins; site of mRNA and tRNA synthesis 7. Smooth endoplasmic reticulum (ER): contains enzymes that synthesize lipids and detoxify certain hydrophobic molecules 8. Rough endoplasmic reticulum (ER): functions in the synthesis, processing, and sorting of secreted proteins, lysosomal proteins, and certain membrane proteins RIBOSOMES

Antibody labeling of cell components (Indirect Immuno-fluorescence Microscopy)

1. Recall western blotting 2. Secondary antibody tagged with a fluorescent dye (goat anti-rabbit) 3. Primary antibody (from rabbit) against lamin 4. Antigen within the cell (ex: lamin A protein: Progeria) 5. Fluorescent dyes commonly used: Rhodamine (red), Fluorescein (green), Cyan (orange) 6. Multiple dyes → multiple antibodies → multiple antigens

Phase Contrast Microscopy:

1. Waves are in phase, so they combine - constructive 2. Waves are out of phase, so they cancel each other - destructive