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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)
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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
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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
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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
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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)
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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
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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 degradationTags and removes only the N-terminus AA
- Repeated trials determine successive AA in order
- ALTERNATE METHODS
- mass spectrometry
- DNA sequencing of coding gene
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Compare and contrast Edmund degradation and Amino terminus determination
- PURPOSE
- Edmund - get sequence of AA
- ATD - determine AA @ N-terminus
- CHEMICALSEdmund - 1) phenylisothiocyanate 2)CF3COOH
- ATD - 6M HCl, 100C, overnight
- PRODUCTS
- Edmund - 1 AA-PTC adduct and rest of peptide
- ATD - 1 AA-aromatic adduct and all other untagged AA
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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
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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
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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)
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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
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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
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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
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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
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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)
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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
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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
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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
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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)
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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
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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)
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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
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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
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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
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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)
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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]
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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
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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
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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
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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
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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
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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)
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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
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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
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