c430 final exam amino acids, proteins

  1. 1. Draw how a peptide bond is formed. What type of reaction is this?

    2. How are proteins read?

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    2. Amino terminus ---- carboxy terminus
  2. 1. What are three characteristics of the peptide bond?

    2. Which is more stable: Cis/trans form of peptide bond? Why?

    3. Can you freely rotate around the peptide bond?

    4. How can peptide bond rotate?
    1. Trans, planar, 40% double bond character due to resonance seen in C-N.

    2. Trans (10 KJ more stable) because it's less likely to have steric hindrance.

    3. No.

    4. Only around a-carbons (think of a-carbons as hinge points), all four atoms in an amino acid travel together in a plane.
  3. 1. How many bonds separate sequential a-carbons in polypeptide chain?

    2. Which two bonds can rotate? Three names for each.

    3. What determines whether the above bonds can rotate?

    4. Why else might other single bonds in backbone also be rotationally hindered? (2)

    5. Which bond is never ever free to rotate?
    1. 3

    2. N-aC and aC-C (Image Upload 2 and Image Upload 3) or (torsiol & dihedral) bonds.

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    3. Sterics

    4. Size and/or charge of the R group

    5. C-N bond
  4. 1. What tells us which combos of phi and psi are allowed?

    2. Are there more allowed or unallowed combos? Why?

    3. What is each graph specific for?

    4. What does dark blue correspond to?

    5. Medium blue? Light blue?

    6. Tan?

    7. What structure has the same combos of phi and psi?
    • 1. Ramachandran plots
    • 2. Unallowed b/c of sterics
    • 3. A specific enantiomer of an amino acid (e.g., L-alanine)

    4. No steric overlap and fully allowed

    5. Medium blue? conformations allowed at extreme limits for unfavorable atomic contacts

    Light blue - conformations that are permissible if a little flexibility is allowed in dihedral angles

    6. Tan - disallowed combos b/c it is sterically unfavorable and thermodynamically impossible

    7. A-helices
  5. 1. Why can't you have both phi and psi be at 0 degrees ever?

    2. Do unbranched side chains for other L - residues look identical to alanines?

    3. What can't those with PKU eat?
    1. Steric interference

    2. yes.

    3. Synthetic AA aspartame or phenylalanine, b/c they're missing phenylhydroxylase which turns phenylalanine into tyrosine.
  6. 1. What are the levels of structural organization for proteins? Define each broadly.

    2. What is similar about the tertiary & quaternary structure?
    1. Linear sequence of AA, dictates protein function & determines folding of protein.

    2. 3D path in space of amide backbone (doesn't consider contribution of R groups) - primary for stabilizing structure

    3. 3D folding of protein w/ contributions of R groups included. R groups help bring in whole structure

    4. Optional - composed of more than one polypeptide subunit (i.e., Hb)

    2. same forces hold tertiary and quaternary structures together
  7. 1. Which structure dictates all following structures?

    1. Which structure makes each protein unique? What limits the 20^n number of different combinations?

    2. What stabilizes the secondary structure?

    3. How do are R groups organized in relation to backbone of secondary structure?

    4. What leads to particularly stable structures?

    5. How many AA residues per turn of a-helix?

    6. Who hydrogen bonds with who?
    • 1. Primary, sterics (ramachandran plot)
    • 2. H bonding
    • 3. Point out and away from backbone
    • 4. Contiguous phi and psi angles that are identical lead to a-helix and b-sheet

    5. 3.4 residues/turn

    6. n and n+4
  9. 1. What is the beta sheet stabilized by? Where? Is it contiguous?

    2. What does parallel beta sheet look like? Anti-parallel beta sheet?
    1. H-bonding between adjacent chains, but it's not contiguous

  10. What determines tertiary structure? (5)

    Which is the sole strong force?

    3 ions involved?
    1. Disulfide bond (2 cysteine residues) - covalent bond sole strong force -S-S <---> -SH HS- via redox.

    2. Ionic interactions via salt bridges

    3. H-bonding

    4. Hydrophobic interactions

    5. Metallic ion interactions (i.e., Fe2+, Mg2+, Mn2+)
  11. 1. What is a molten globule?
    2. What are general characteristics of a tertiary structure? Why?

    3. What are the stabilizing forces of the quaternary structure?
    • 1. Almost folded protein
    • 2. Hydrophilic exterior and hydrophobic interior thanks to dS
    • 3. Stabilizing forces are the same as tertiary forces (disulfide bonds, ion interactions, H-bonding, hydrophobic, metallic ion)
  12. 1. What drives protein folding? (3)
    2. What is the lowest energy state of the protein?

    3. Is protein folding random or nonrandom? How do we know?

    4. Draw proposed sequence of events in protein folding. Which one is rapidly formed?
    1. Nonpolar R groups on the interior, Polar R groups on exterior, and nonrandom processes that drive organization from primary structure to tertiary (quaternary) structure.

    2. Fodled state

    3. Nonrandom, we know this b/c if we searched for every possible conformation, it would take a REALLY long time. However, it takes much less time, so we know its nonrandom.

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  13. 1. How is protein folding spontaneous?

    2. Why?

    3. From what source?
    1. More likely due to entropy than enthalpy

    2. b/c huge exothermic rxn would denature the protein

    3. ENTHALPY - b/c H2O molecules are released to the outside, away from hydrophobic molecules, there is a net increase in dS, despite new compactness of protein.
  14. 1. How do proteins fold properly? Define.

    2. Why is this necessary?

    3. How does this work? (mech)

    4. Can proteins fold correctly w/o help?
    1. With the use of chaperones. Chaperones are folding catalysts that prevent formation misformed proteins.

    2. Because high [protein] or incorrect folding can lead to aggregation or precipitation, which is bad.

    3. Mechanism:  (1) when proteins fold, there is the exposure of hydrophobic R groups. 

    (2) Chaperones will bind to exposed R groups to facilitate correct folding

    4. Yes.
  15. 1. What causes denaturing? (very broadly) - 3
    2. What can be used to disrupt disulfide bonds?
    3. What causes irreversible denaturation?
    4. What type of denaturing does urea do?
    5. What does it allow?
    • 1. Heat, pH, chaeotropic salts
    • 2. BME
    • 3. Heat (think of freezing a fried egg)
    • 4. Denatures by interacting with R groups, amide backbone interactions,hydrophobic interactions etc that put proteins together.

    5. Urea allowed protein to be denatured but to stay in solution as well
  16. 1. What was the famous experiment by Christian Anfinsen using RNAse A?
    2. How did he denature his globular protein? (2) What was the role of each?

    3. How did he remove them?
    4. What happened when he removed the denaturation agents?  (2)

    5. What are the major points (3)
    1. He wanted to show that denaturation of some proteins was reversible.

    2. Urea (broke up hydrophobic bonds) and reducing agent (broke disulfide bonds)

    3. He removed the urea & reducing agent using dialysis and added in an oxidizing agent

    4. The protein spontaneously refolded into its correct position and had full catalytic actvitiy

    5. (1) protein folding isn't random (2) chaperones aren't rquired (3) as long as you have the prim seq, you can get correct folded structure
  17. 1. What does heat do?
    2. pH?
    3. Chaeotropic salts? (2 examples)
    1. Disrupts weak forces. In a small temp range, protein collapses, meaning that dissolution of one area --> dissolution of another (domino effect). Irreversible!

    2. pH interferes with net charge on protein, causing electrostatic repulsion & H-bond disruption.

    Ex. Lys+| |Asp- salt bridge. IF pH decreases, Asp will be protonated losing salt bridge.

    3. Urea - mainly for hydrophobic interactions - allows protein to be denatured but to stay soluble in solution.

    Ammonium sulfate - allows protein to stay folded, but affects solubility
  18. 1. What do we take advantage of the purify proteins?
    2. What are two types of methods of cell disruption? (1-2, 1-1)

    3. What are protein purification methods based on? (4) What would you use for each category?
    1. Unique characteristics due to unique primary sequence

    2. Mechanical methods (blender/sonication (using high freq sound waves to open cell)

    and Enzymatic methods (lysozyme - naturally occurring hydrolase found in tears that degrade bacterial cell wall).

    3. Solubility - use chaeotropic salts, charge - ion exchange chromatography, size - gel filtration and natural binding affinity.
  19. 1. How can you use pH to purify proteins?

    2. How do you use ionic strength?  What type of salt should be used?

    3. What are the types of chromatography? (3)
    1. Each protein has its own isoelectric point. At that point, the solubility of the molecule is minimized.

    2. Increasing [salt] decreases protein solubility. Add salt, centrifuge lysate to separate precipitate from solution, then continue to add salt, etc. Ammonium sulfate.

    1. Ion exchange, size exclusion, and natural affinity
  20. 1. Describe ion exchange chromatography: what does it take advantage of? What is the matrix and what does it bind?

    2. What are cation exchangers? Anion exchangers?

    3. What does affinity of each protein for the charged groups on the column depend on? (2)

    4. How do you make the final protein elute?
    1. Intrinsic charge of protein (size AND magnitude of charge) - matrix = stationary phase that is a synthetic polymer containing bound charged groups

    2. Cation exchangers are resins with bound anionic groups; anion exchangers have bound cationic groups

    3. pH (which determines ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution

    4. Add excess salt to disrupt electrostatic interactions
  21. 1. What is gel filtration chromatography good for?
    1. Separating proteins w/ very large difference in molecular weight. Otherwise use SDS page.
  22. 1. What elutes out of gel filtration/size exclusion chromatography first?

    2. How do you collect proteins of interest from natural binding affinity chromatography? (2)

    3. What affects how protein will move in electric field through SDS Page? (3)

    4. What does SDS confer on all proteins? (2) How? What does it separate everything based on?

    5. How are proteins later visualized?

    6. Do smaller or larger proteins move farther?

    7. What does SDS leave intact?
    • 1. Large proteins
    • 2. Excess salt/excess free ligand solution

    3. Size, shape, and charge

    4. Same shape (linear) & charge:mass ratio.

    Shape - SDS breaks all noncovalent bonds, denaturing protein into its linear form.

    Charge - SDS is highly negative, binds all around protein, making it super negative. Separates everything based on MW.

    5. Coommassie blue

    6. Smaller

    7. Disulfide bonds (b/c they're covalent)
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c430 final exam amino acids, proteins