Exam 2 - Mods 4 - 6

Mod 4

Protein biochemistry methods and hemoglobin structure

Compare and contrast X ray crystallography and NMR spectroscopy as tools for determining protein structure

X ray crystallography - determines position of all atoms in protein crystal using diffraction patterns of x rays projected onto the crystal. Great for determining 3D structure of larger proteins, however difficult to obtain a very pure, isolated specimen of target protein. NMR spectroscopy - determines relative position and atom connectivity based on nuclear spin properties in magnetic field; NMR spectroscopy uses soluble proteins at high concentrations. *X ray diffraction works with larger proteins, however protein crystals are difficult to obtain; NMR spectroscopy can use proteins in solution, however it can only be effectively used to study smaller proteins*

Identify the mutation and resulting structural change in HbS that produces sickle cell anemia

Mutation that results in sickle cell anemia is a change in glutamic acid 6 into a valine. This results in an extra hydrophobic patch on the beta subunits of Hb; these hydrophobic patches can attach to each other via van der waal forces, which results in aggregation of HbS into long chains that distorts the shape of the RBC (causes the RBC to have a sickled shape). *BetaS Val6 can interact with BetaS Leucine88 and BetaS-phenylalanine 85 to produce long chains, aggregates, of HbS*

Describe the structure and function of myoglobin and hemoglobin

Myoglobin is monomeric (has only 1 subunit) protein that serves as an oxygen storage protein in tissues (such as skeletal muscle), whereas hemoglobin is a tetrameric (4 subunits) protein that transports oxygen from lungs to tissues. Myoglobin and Hb are both heme utilizing O2 binding proteins, and each subunit consist of *8 alpha helices* with a heme group attached to each. Important to note that the structure of Mb and Hb are very similar, however the amino acid sequence for the subunits are actually *very low* in similarity.

Identify the key steps involved in isolating proteins from whole cells

1) Centrifugation - separates macromolecules on the basis of density, centrifugal force, and centrifugation time (results in different layers of cellular components.; may have a layer of nuclei, mitochondria, components of the plasma membrane, and the cytosol) 2) Column Chromatography - umbrella term for a variety of methods to separate target protein from bulk (unwanted) protein. Includes: a) Gel filtration b) ion exchange c) affinity chromatography d) isoelectric focusing e) 2D SDS PAGE *Can use a combo of different column chromatography to isolate proteins; the first three are usually used to separate large amounts of proteins, and the last two are used to separate small amounts of protein 3) Once separated initial mixture of whole cells to its individual components, then using column chromatography to separate target protein from bulk protein, the *specific activity* of different fractions can be calculated to identify the fraction with the greatest amount of target protein. *specific activity* = amount/activity of target protein/total amount of protein

Define the different types of column chromatography

1) Gel filtration chromatography - uses porous carbohydrate beads made of acrylic that separate proteins on the basis of *size* Larger proteins will pass through the gel matrix *first* while smaller proteins will pass through the gel matrix *last* (will get stuck in tight spaces in porous carbohydrate beads) 2) Ion exchange chromatography - uses charge differences to separate target protein from bulk protein. *Anion exchange resin ATTRACTS anions and is positively charged!!* *Cation exchange resin ATTRACTS cations and is negatively charged!!* 3) Affinity chromatography - exploits specific binding properties of the target protein to separate it from other cellular proteins lacking a binding site. Receptor for target protein may line the gel matrix; non target proteins will likely pass through first, and target protein passes through last (since it will bind to its receptors in the gel matrix)

Describe 5 major functional classes of proteins in cells and provide a representative example of each

1) Metabolic enzymes - chemical catalysts that decrease activation energy of a reaction without changing equilibrium constant or standard change in free energy (*only affects the rate of a reaction). Ex - malate dehydrogenase, metabolic enzyme that speeds conversion of malate into oxaloacetate 2) Structural proteins - often assembled into polymers to form filaments that provide cell structure and facilitate movement Ex - actin and tubulin (actin are often arranged into long protein fibers, observed to make thin filaments which can be used in muscle contraction, controlling cell shape and migration) 3) Transport proteins - function as selective pores to permit or transport polar metabolites across the membrane (nonpolar lipid bilayer) Ex - Ca2+ ATPase transporter proteins uses ATP hydrolysis to pump Ca2+ ions across cell membranes against their concentration gradient 4) Cell signaling proteins - respond to changes in environment and relay information about these changes to other proteins. Ex - Erythropoietin receptor which is an integral membrane receptor with two subunits, which associate into a homodimer to form a single hormone binding site. 5) Genome caretaker proteins - maintain chemical integrity of genetic information stored in DNA and RNA Ex - bacterial RcBCD protein complex binds to DNA, unwinds the DNA helix, and cleaves one of the 2 strands to facilitate DNA repair or recombination

State the three assumptions made when using Michaelis-Menten kinetics and define the Michaelis constant

1) Reaction is analyzed at a time when NO appreciable product has been generated (so the back reaction of ES forming EP with a rate constant of k-2 is negligible) 2) Product released is assumed to be a rapid step in the process, so the conversion of EP to E + P does not need to be explicitly considered. 3) The steady state condition is reached quickly, such that [ES] is relatively constant after an initial reaction time. By working under conditions where [S] >>> [E], the [ES] remains approximately constant.

Summarize the separation methods that can be combined in 2D PAGE

2D gel electrophoresis combines both *isoelectric focusing (separation of proteins on the basis of charge and pH)* and SDS PAGE. In isoelectric focusing, proteins migrate toward oppositely charged ends (a cathode which has a negative charge or an anode which has a positive charge) until a point in the pH gradient where they have n net charge (the pI point, the point at which the protein has no charge). In 2D PAGE, proteins can be separated on the bases of isoelectric focusing first (based on charge and pH); then, these same proteins are run through SDS PAGE in the other direction. Results in a 2D gel; one axis represents proteins separated on pI (isoelectric point), and the other represents proteins separated on the bases of mass.

List three major ways that enzymes increase the rate of a reaction and the three most common reaction mechanisms in enzyme active sites

3 major ways that enzymes increase the rate of a reaction: 1) stabilize the transition state (by noncovalent interactions between the substrate and the enzyme) 2) provide alternate path for product formation, formation of a stable environment 3) Optimal substrate orientation and increased local concentration 3 most common reaction mechanisms in enzyme active sites: 1) coenzyme dependent redox reactions 2) metabolic transformation reactions 3) reversible covalent modification reactions

List the amino acids that compose the catalytic triad of chymotrypsin and describe the six steps in the chymotrypsin reaction mechanism

Catalytic triad of chymotrypsin: His57, Asp102, Ser195 1) Polypeptide substrate binds to enzyme active site 2) His 57 removes a proton from Serine 195, which allows nucleophilic attack by the serine oxygen on the carbonyl carbon of the peptide 3) His 57 donates a proton to the amine group of the substrate, resulting in peptide bond cleavage. *Carboxy terminal fragment is released as the first product* *this completes the first phase, steps 1-3, a covalent acyl enzyme intermediate is formed between Ser 195 and the polypeptide, which promotes cleavage and the carboxy term end of the peptide is released* 4) Water enters active site, His57 acts as a general base and removes proton from H2O. Results in OH-, which acts as a nucleophile and attacks the carbonyl carbon of the covalent acyl enzyme intermediate. 5) His57 donates a proton to Ser195, resulting in cleavage of the acyl enzyme intermediate (pt of substrate that remain in chymotrypsin). The amino term fragment is released as the second product, and the catalytic triad is regenerated. 6) The functional catalytic triad is regenerated within the enzyme active site.

Interpret the results of protease treatment followed by Edman degradation to determine the sequence of a peptide

Edman degradation is a method to sequence short polypeptides one amino acid at a time. Polypeptide is first labeled at the N term, with PITC; acid hydrolysis then follows to yield PIH amino acid derivative, which can then be identified using high performance liquid chromatography (HPLC). 20 standard known amino acids can be run through HPLC first, and each unknown amino acid cleaved from the polypeptide can be run through HPLC and have their results compared to that of the standard amino acids to identify the sequence of the short polypeptide. Differential protein cleavage (process where a larger protein is cleaved into shorter fragments) is used to generate small overlapping polypeptides that can be sequenced by edman degradation one amino acid at a time. Example: Use the following information to determine the polypeptide sequence: Trypsin digest: LMKK, MGTCE, WDER Chymotrypsin digest: CE, LMYKW, DERMGF Result: LMYKWDERMGFCE

Mod 6

Enzymes: Function, reaction mechanisms, and kinetics

Identify structural differences between adult and fetal hemoglobin that impact 2,3-BPG association

Fetal hemoglobin (alpha(2)gamma(2) has a decreased affinity for the negative effector 2,3-BPG due to the histidine 143S difference in the gamma subunit. As a result, fetal Hb is shifted to the R state (relaxed state, has greater affinity for oxygen than adult Hb).

Explain the migration process of gel electrophoresis and how marker proteins are used to estimate the apparent molecular mass of an unknown protein

Gel electrophoresis, such as polyacrylamide gel electrophoresis (PAGE) uses a solid polyacrylamide gel to separate proteins on the basis of both *size and charge.* SDS is a compound that is exposed to protein mixture; disrupts secondary protein structure and uniformly binds to hydrophobic regions of every protein regardless of mass or size. Results in all proteins in a protein mixtures to have a uniform negative charge. Larger proteins will migrate over less distance in a SDS PAGE gel, whereas smaller proteins will migrate over a longer distance in the same gel. Thus, larger proteins separated by SDS PAGE gel electrophoresis is done with a *lower* percentage of polyacrylamide, while smaller proteins are separated wit ha *higher* percentage of polyacrylamide. Marker proteins, or proteins of known molecular mass, can be run through an SDS PAGE gel in a lane adjacent to unknown proteins from a sample. The migration distance of the marker proteins with known molecular masses can be compared to that of the unknown proteins to estimate their apparent molecular mass.

Module 5

Hemoglobin allostery, transport proteins, and actin-myosin

Analyze the structural changes that occur when oxygen binds to the heme group

Proximal histidine, HisF8, located on the F helix, coordinates with iron in the heme group. Distal histidine, HisE7, located on the E helix, forms a hydrogen bond with oxygen and stabilizes its interaction with the heme group. In the absence of oxygen, the heme group is puckered since the diameter of the iron atom is large. Upon oxygen binding, shared electrons result in slightly smaller atomic radius for iron, resulting in flattening of the heme group; *movement of iron into the same plane of the porphyrin ring results in the F helix being pulled towards the proximal histidine*

Outline the steps of solid-phase peptide synthesis

Solid phase peptide synthesis - process of making a short polypeptide by using blocking and reactive groups C term of amino acid is attached to a resin and the N term blocking group Fmoc is removed by treatment with base. A covalent linkage is then formed between DIC-activated carboxyl group and Fmoc-blocked amino acid. Outline of steps of solid phase peptide synthesis: 1) Deblocking of residue 1 attached to resin (removal of Fmoc from amino group of residue 1) 2) Activation of Fmoc blocked residue 2 (addition of DDC at the carboxy end of the second amino acid) 3) Coupling of amino acids by creating a peptide bond between the deblocked amino group of residue 1 and the activated carboxy group of residue 2 In the very final step, the *protecting groups are removed,* and the polypeptide is released from the matrix. This newly made oligopeptide can then act as an antigen to provide certain antibodies of interest for study (can inject peptide into a specimen, specimen produces antibodies of interest for study).