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2013-10-25 09:27:37

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  1. What are the ionizable AA w/ pKr and charge at pH7
    • *DR CHEKY
    • Arginine (R): pKr 12.48 (+1 @ pH7)
    • Aspartate (D): pKr 3.65 (-1 @ pH7)
    • Glutamate (E): pKr 4.25 (-1 @ pH7)
    • Lysine (K): pKr 10.53 (+1 @ pH7)
    • Histidine (H): pKr 6.00 (neutral @ pH7)
    • Cysteine (C): pKr 8.18 (0 @ pH7)
    • Tyrosine (Y): pKr 10.07 (0 @ pH7)
  2. What is the general process for preparing a sample for protein analysis?
    • Crude extract: lysing of tissue/microbial cells in soln
    • Differential centrifugation: separate by size to organelle level
    • fractionation: separate proteins by size or charge
    • Selective precipitiaon using NH4SO4, base, or acid)
    • Chromatography
    • dialysis: purification of above soln that leaves on purified protein
  3. Liquid Chromatography brief explanation, what is stationary and mobile phase?
    • Used to separate a mixture based on IMF
    • Analyte is analyzed in gas form (no IMF in gases)
    • Stationary: porous solid material w/ desired properties in column
    • mobile: buffered soln moved through the stationary phase
    • The protein soln moves w/ mobile phase through the column
  4. Types of chromoatography with brief description
    • Size exclusion: stationary phase is polymeric beads with specifically sized holes
    • large molecules elute first (too big to interact)
    • smaller = more interaction = last to elute
    • Cation exchange: stationary phase has - charge
    • more positive = more interaction = last to elute
    • Anion exchange: stationary phase has + charge
    • more negative = more interaction = last to elute
    • Affinity: higher affinity to ligand on stationary phase = more interaction = last to elute
    • often antibodies
    • HPLC: small, tightly packed, tubes ↑ SA and pressure
  5. Electrophoresis brief explanation WITH VARIOUS TYPES AND DESCRIPTION
    • Used to separate proteins by size and pI
    • PAGE (polyacrylamide gel electrophoresis: has a voltage gradient that causes AA to move based on size and SA (folding, etc)
    • SDS (sodium dodecycl sulfate): added to PAGE
    • interacts with peptide backbone causing denaturation (negates SA separation) AND adding 2 neg charges per AA (negates charge separation)
    • SDS-PAGE: only factor causing movement is MW (smallest MW = furthest movement)
    • Isoelectric focusing: PAGE + ampholyte (acid or base)
    • uses pH gradient to determine pI
    • When pI is reached protein stops moving (no charge = no force for movement)
    • 2D gel electrophoresis:
    • 1st dimension - isoelctric focusing
    • 2nd dimension - SDS page (MW separation)
  6. What are the specific enzymes used for cleaving peptides and where do they cut?
    • Always on peptide bond
    • Trypsin: C side of K, R
    • Chymotrypsin: C side of F, W, Y
    • V8 Protease: C side of D, E
    • Pepsin: N side of L, F, W, Y
    • CNBr (cyanogen bromide): C side of M
  7. Describe the steps involved in determining the primary structure of a protein (do not explain alternate methods in detail)
    • Determine amount of each AA present
    • 1.6M HCl, 100C, overnight --> individual AA
    • 2. dabsylchloride or dansylchlorie --> detectable adduct for each AA
    • Determine the amino terminus
    • 1. 1-fluoro-2,4-dinitrobenzene --> tagged amino terminus (interacts with free amino end)
    • 2. 6M HCl, 100C, overnight --> tagged AA + other AA
    • Reduce disulfide linkages
    • Only necessary if there are 2 Cys in peptide
    • Dithiothreitol then Iodoacetate, *performic acid, TCEP (best)
    • *performic acid will oxidize W
    • Cleave peptides into smaller peptides
    • Trypsin, Chymotrypsin, V8 Protease, Pepsin, CNBr
    • Edmund degradation
    • Tags and removes only the N-terminus AA
    • Repeated trials determine successive AA in order
    • mass spectrometry
    • DNA sequencing of coding gene
  8. Compare and contrast Edmund degradation and Amino terminus determination
    • Edmund - get sequence of AA
    • ATD - determine AA @ N-terminus
    • Edmund - 1) phenylisothiocyanate 2)CF3COOH
    • ATD - 6M HCl, 100C, overnight
    • Edmund - 1 AA-PTC adduct and rest of peptide
    • ATD - 1 AA-aromatic adduct and all other untagged AA
  9. What is mass spectrometry? Describe the use of mass spectrometry in determining protein structure
    • Mass spec analyzes charged gaseous molecules
    • challenge - GETTING charged gaseous proteins
    • Electrospray: liquid protein soln passed through tiny capillary with voltage added, then passed through to magnet area
    • Magnet determines # of charges and abundance
    • MS-MS can give accurate sequence
    • MALDI-TOF: focused, high E laser lifts protein from solvent (soln w/ many weak acids or bases)
    • TOF (time of flight) determines how long the charged molecule stays in MS
  10. What are the folding rules that govern tertiary structure of a protein?  Why? Which are most important?  Which AA would apply to them?
    • disulfide linkages (covalent)
    • C
    • Hydrophobic inside (decrease entropy of bulk water)
    • F, I, V, L, W, P, A, G, T, M, C
    • H-bonding/polar side chains (strongly destabilizing unless they are partnered)
    • S, T, Q, N
    • salt bridge (can be stabilizing +/- or destabilizing -/-)
    • E,D (acidic)
    • R, K (basic) potentially H
  11. Describe the α-helix, what types of interactions, any special AA?
    • secondary structure
    • right handed helix
    • 3.6 AA per turn (#1 interacts w/ #4)
    • AA on outside, backbone atoms fill "inside"
    • stabilized by 1,4 interactions...
    • 1. H-bonding of backbone (C=O and NH2)
    • 2. Salt bridge
    • 3. H-bonding
    • 4. stacked aromatics
    • 5. hydrophobic groups
    • NO PROLINE (too rigid, incorrect bond angle)
    • NO GLYCINE (too flexible)
    • NO 1,4 CYSTEINES (bind and ruin angle)
    • ex - keratin (hair, skin, nails, etc)
  12. Describe the β-sheet, what types of interactions, any special AA?
    • Extended structures, very long/thin compared to helices
    • Allows a huge amount of H-bonding between backbone in sheets (very strong)
    • can be parallel (terminuses of sheets are on same side) or antiparallel
    • only small R groups (A, G)
    • examples - silk, wool, spiderweb
  13. Describe the α chain, what types of interactions, any special AA?
    • left handed helix
    • 3AA/turn
    • Typical structure is G - X - Y (X is typically P and Y is typically hydroxyproline BUT X/Y an be V, A, H, or K)
    • HYDROXYPROLINE is of utmost important for the covalent cross-linkages within procollagen and between collagen fibers
    • huge amounts of G (very tight turns)
    • ex-collagen
  14. Describe the β-turn, what types of interactions, any special AA?
    • 2 types - I w/ P in position 2, and II w/ G in position 3
    • Both types stabilize the turn for H bonding between C=O and NH2 of AA1,4
    • Typically link β-sheets OR provide link between β sheets and α helices
    • 180degree turn involving 4 AA res
    • G COMMON due to being small and very flexible
    • cis P COMMON to stabilize the turn
    • *conversion from trans P to cis P requires a large amount of E
    • **99.95% P in peptides is trans, the .05 cis is in β-turns
  15. Describe keratin (Function, structure, found in)
    • Function is protection
    • 2 α-helices (right handed) form a left handed coiled coil
    • These arrange to form fibrils, etc (lego bricks) which have disulfide linkages between some of them (more disulfide links = more strength/harder)
    • Found in horns, nails, hooves, hair, skin
  16. Describe collagen (Function, structure, found in)
    • Function is structural support (acts as "rebar" in bones that CaCO3 and Ca2(PO4)2 bind to)
    • 3 α-chains (left handed) form right-handed coiled coil w/ G at intersections of α chains
    • Staggered conformation (lego bricks) like Keratin gives great strength
    • ~27 different human forms of collagen
    • 1/3 is G (α-chains) due to tight turns
    • HYDROXYPROLINE is important to cross linking and structure
    • AA in general have non-bulky sidechains (hydroxylysine is bulkiest)
    • MAJOR PROTEIN IN BODIES (extracellular matrix)
  17. List the ways the 4-hydroxyproline is important to collagen
    • 1. Exo formation needed for Y position (G-X-Y) so α chains can form triple helix
    • (exo formation only occurs after hydroxylation)
    • 2. Forms the cross links in collagen fibers that give it its strength
    • 3. Upregulation of gene expression for collagen synthesis
  18. How is hydroxyproline affected by Vitamin C? Discuss Vit C deficiency
    • Hydroxylation of P requires Fe2+
    • Fe2+ is regenerated by Vit C oxidation
    • Vitamin C deficiency = no hydroxyproline = defective collagen resulting in...
    • bleeding gums, bruises, broken bones, death
  19. Describe elastin (Function, structure, found in)
    • α-helical character gives elastic properties (lungs, skin, blood vessels)
    • In lungs - allows expansion of chest cavity and compression for breathing
    • covalent linkages
  20. Cytosolic protein folding and WHY (in relation to free energy)
    • ΔG=ΔH-TΔS
    • I. Hydrophobic inside (+ΔS) - increase entropy of bulk water
    • II. Hydrophillic outside (-ΔH) - decrease enthalpy by creating H-bonds with water
    • maximize disulfide linkages (covalent)
    • form salt bridge, inside OR outside (ionic)
    • form H-bonds, inside or outside (H-bonding)
    • induced dipole-induced dipole (hydrophobic)
  21. Describe the methods for examining 3D protein structure, including benefits and disadvantages and medical use
    • X-Ray crystallography: measures the diffraction pattern of X-rays sent at the crystal structure
    • advantages - can compare to protein database via computer work
    • disadvantages - need a crystal (especially difficult for membrane-bound proteins), crystals are static so no dynamic processes can occur
    • In hospital - X-rays pick up only dense material (bone)
    • NMR: takes advantage of nuclear spin.  Either spins w/ magnet (lower E) or against magnet if provided an energy pulse (high E).  The difference between the lowE and highE states can be quantified (E=hν)
    • advantages - solution allows for dynamic activity to be captured (enzyme-substrate binding, inhibitors, etc)
    • in hospital - MRIs give high resolution to soft tissue
  22. What aids protein folding (besides AA sequences) w/ details
    • Heat shock proteins (Hsp70 and Hsp40): bind proteins (using ATP) to prevent hydrophobic R groups from aggregating
    • *doesn't fold... holds in specific manner
    • Chaperonins: large, multisubunit assembly that uses ATP to fold proteins
    • *actually performs folding
    • PDI (Protein disulfide isomerase): enzyme that shuffles the disulfide linkages in a protein (reduce, reorganize, oxidize)
    • PPI (Peptide prolyl cis-trans isomerase): enzyme that converts trans proline (99.95%) to cis proline (.05%) (required for β turns)
  23. What are the diseases caused by improper protein folding?
    • Alzheimers, Parkinson's, Huntington's - neurological
    • Type II diabetes - pancreas damage
    • Amylodosis - kidney and liver damage
    • Improper folding causes the single, soluble proteins to aggregate into large fibrous masses (a slight misfolding allows the β sheets to find eachother, and the hydrophobic interactions are so strong they "take over")
    • Prions - an infectious protein.  PrPC consists of 3 helices, while PrPSC consists of 2 helices and beta sheets! The beta sheets conglomerate into amyloid plaques
  24. Compare/contrast myoglobin and hemoglobin (general)
    • Myoglobin: 1 subunit, 1 heme group, 1 Fe2+ is bound to heme
    • Functions to store O2 in muscle tissues
    • Hemoglobin: 4 subunits (HbA = 2α and 2β), 4 heme groups, 1 Fe2+ is bound to each heme (4 total)
    • Functions to transport O2 from lungs to tissues
  25. What accounts for the difference in function between myoglobin and hemoglobin?
    • Myoglobin has 1 subunit: as PO2 rises the binding of O2 to myoglobin to a saturation value
    • Hemoglobin has 4 subunits: as PO2 rises the various arrangements of subunit interaction give various "forms"
    • "R form" in the lungs which provides a high affinity of O2 binding
    • "T form" in the tissue which has a low affinity for O2
  26. Describe how hemoglobin alternates between T and R forms
    • Cooperativity of the subunits
    • When O2 binds to 1 subunit the shape alters and causes neighboring subunits to increase their O2 affinity
    • (seen as an an equilibrium shift below)
    • TTTT <--O2> TTTR <--O2-> TTRR <-O2--> TRRR <O2---> RRRR
    • The reaction initially occurs through Le Chatlier (disfavored, but huge amount of O2 in lungs shifts equilibrium to the otherside)
  27. Describe protein-ligand interactions quantitatively with equations
    • P + L ⇌ PL
    • (association) Ka=[PL]/[P][L] ; ratea=ka[P][L]
    • 2nd order (2 reactants) ∴ ka units are M-1s-1
    • (dissociation) Kd=[P][L]/[PL] ; rated=kd[PL]
    • 1st order (1 reactant) ∴ ka units are s-1
    • At equilibrium ratea=rated ; ka[P][L]=kd[D] ; Ka/Kd = [PL]/[P][L]
  28. What is θ both theoretically and mathematically?
    • θ = binding sites occupied / total binding sites ∴ θ = [PL]/([PL]+[P])
    • After subs -> θ=
    • *dissociation constant NOT rate constant
    • **important - When [L] = Kd = 1/Ka then θ = .5
    • *dissociation constant NOT rate constant
  29. What are the stabilizing factors of the T form of hemoglobin?  What causes it to shift to R form? Be detailed.
    • T form: salt bridgest between R groups at the terminal ends of globins
    • ionic attractions between 2,3-bisphosphoglycerate and Lys/Arg
    • The binding of O2 to the heme iron causes iron to move out of the heme plane, which causes movement of helix F and breakage of the salt bridges (a stabilizing factor for "T")
    • Lack of stabilization leads to a shift in equilibrium toward the R form
  30. Describe the Bohr effect
    • At low pH the HbA affinity for CO2 and H+ increases and the affinity for O2 decreases (the tissues)
    • At high pH the HgA affinity for CO2 and H+ decreases and the affinity for O2 increases (the lungs)
    • *It can be noted that cooperativity exists for O2 binding to Fe on heme AND for H+ binding to hemoglobin
    • summary - (1) high CO2 and (2) Low pH stabilize the T form and decrease O2 affinity
  31. What is Histidine HC3 and explain it in detail
    • A major contributor to the Bohr effect
    • When protonated, His HC3 forms one of the salt bridges required to stabilize the T form of hemoglobin.  This stabilization allows His HC3 to have an abnormally high pKr value in the T form.
    • In the R state this salt bridge cannot form, and the pKr value reverts to it's normal state (6.00)
    • **As [H+] rises, protonation of His HC3 promotes release of O2 by favoring a transition to the T state
  32. How is CO2 transported in the body?
    • 1. The major blood buffer CO2 + H2O <-> H2CO3 <->HCO3- + H3O+
    • 2. In the form of a carbamate group at the N-terminus of each globin chain
  33. What is BPG and how does it work?
    • 2,3-bisphosphoglycerate: greatly reduces (and therefore regulates) the O2 binding affinity of hemoglobin (stabilizing T state, "forced out" of R state).
    • Important in the adaption to lower pO2 at higher altitudes (body must produce more BPG to deliver more O2 to tissues)
    • More BPG = less O2 affinity = more O2 delivered to tissues (doesn't affect R state in lungs, so O2 "gathering" is unaffected)
  34. Describe the role of BPG in fetal hemoglobin
    • HbF is comprised of γ subunits in place of β subunits
    • This tetramer has a lower affinity for BPG and thus a higher affinity for O2
    • This higher affinity for O2 allows the fetus to "extract" O2 from its mother's blood
  35. Give all information about sickle-cell anemia (it's a lot)
    • A disease that is resultant from mutant hemoglobin (E6V) (glutamate is now valine
    • HbS (α2S2) in place of HbA (α2β2)
    • changing from an aliphatic to a charged residue make a huge structural difference and the HbS's S subunits have hodrophobic pockets in the T form (R forms are very similar)
    • if [HbS] is high then it will polymerize at the hydrophobic pockets.
    • For Those with sickle cell this will result in RBC sickling after exertion (deoxygenation of blood AKA lots of T state) and mass RBC lysis
    • For those with the trait there is not enough HbS to cause sickling