There are 25 questions in all. Questions 1-5 are essays and problems worth 10 points each. Questions 6-25   are multiple choice and are each worth 3 points.

These are constants you may find helpful: R = 8.314 J/mole-K; T = 298 K

1. The five molecules listed in the table can all be used for preparing buffers.

a. Which molecule and its conjugate base would be the best choice to use in preparing a buffer for pH 5.3?

 CH₃COOH (acetic acid)
You did not need to write the conjugate base, as that is given by specifying the acid.

b. What should be the ratio of conjugate base to acid for the buffer?

5.3 = 4.76 + log ratio
0.54 = log ratio

ratio = 3.47 = 3.5

c. In order to have a total concentration of 0.30 M, the acid concentration should be          

and the conjugate base concentration should be                .

ratio = 3.5, meaning that [A^-] = 3.5 [HA]. Also [A^-] + [HA] = 0.30 M
3.5 [HA] + [HA] = 0.30 M
4.5 [HA] = 0.30 M
[HA] = 0.067 M, and [A^-] = 0.233 M

d. Use the Henderson-Hasselbalch equation to show why HCO₃^- and its conjugate base would not be a good choice for this pH.

The pK_a for HCO₃^- = 10.2, so 5.3 = 10.2 + log ratio
- 4.9 = log ratio
ratio = 1.3 x 10^-5, which means there's effectively no conjugate base present at this pH.

2. Draw the peptide that has the sequence V–E–R–Y at pH 7.0 . The pK_a's for the functional groups are given in the table.

3. Dansyl chloride, PITC (phenylisothiocyanate), HCl (aq ), TFA (trifluoracetic acid), and trypsin are all involved in protein sequencing.
a. Choose three and do two things with your choices:
     Explain why each is needed (what each does).
     Put them in the order in which they are used for sequencing a protein.
b. Some proteins must also be treated with dithiothreitol followed by iodoacetate. Why?

     a. I'm showing all possible answers for this.

     b. Dithiothreitol reduces disulfide bonds (–S–S– → 2 –SH), and iodoacetate modifies them so that they don't re-form. All you had to say was that this is required to break disulfide bonds.

4. Recommended: Read the entire question before you start to answer it.
Myoglobin (Mb) was the first protein whose structure and function we studied.
a. Choose one level of protein structure, define it, and briefly (one sentence) describe it
     for myoglobin.
b. Define θ and K_d for myoglobin. How are these two terms related?
     Explain the relationship by writing an equation or by describing it.
c. Name one characteristic from your answers to a and b that is different for hemoglobin
    and state the difference.

a. These are all the possible answers. Only one row was required.

b. θ = the % of binding sites occupied. Note: This is NOT the same as % of O₂ bound.
K_d = the dissociation constant, = ([L] [P])/[PL]
Relationship: θ = [L]/(K_d + [L]) = pO₂/(K_d + pO₂) = pO₂/(P₅₀ + pO 2 )
    Any one of the three was a satisfactory answer.
For K_d, adding a + sign between [L] and [P] changes the meaning.

c. The simplest, easiest answer is to say that hemoglobin has quaternary structure. Most people, though, had started by describing one of the other levels of myoglobin structure, which meant that now they had to introduce something new in order to answer for hemoglobin. This was the reason for the recommendation, so that you could plan ahead and not get into this hole.

Several showed the modified K_D involving a coefficient for L, and that is also fine, and a few people actually drew a small hyperbolic curve for θ in b and then showed a sigmoidal one for hemoglobin in c, which is also fine.

5. Use your peptide from question 2 and answer the questions.

     The form shown is most common from pH                            to pH                             .

    The pI for the peptide is                   .

If this peptide were, instead, a segment of a large globular protein, would you expect it to be on the exterior or the interior of the protein? Explain. Your explanation should include one or more weak interactions involving parts of the segment.

For all peptides, the first pK a is for a side chain and the second for the N-terminal amine.

All peptides were shown with a net charge = 0, so the pI was always the average of the two pK_a's.

Most of these are sequences involving primarily polar and/or charged side chains, so it's easier to answer for the exterior. However, the explanation was more important than the choice. That is, did you specify interactions between side chains and water, which would be appropriate for exterior segments? Naming specific side chains with interactions is a better answer than just naming interactions. For example, glutamate, arginine, and tyrosine all have side chains that can form a hydrogen bond with a water molecule. Or it would be reasonable to say that valine and tyrosine could have hydrophobic interactions with other nonpolar side chains in the interior of the protein.

Use the diagrams for the specified questions (that is, diagram 6-7 for questions 6-7).

       D       6. The molecule shown is a modified                              residue.
(A) valine; (B) serine; (C) isoleucine; (D) threonine.

       D       7. What would be the effect on a protein of this modification? The protein would
(A) be denatured; (B) be unchanged;
(C) lose only its primary structure; (D) change its conformation.

       B       8. Which letter (A-E) labels an atom that could be a hydrogen acceptor for a hydrogen bond that stabilizes secondary structure?

       D       9. The group labeled D could                            which stabilizes                     structure.
(A) accept H⁺ and be attracted to COO^- / tertiary;
(B) donate H⁺ and be attracted to NH₃⁺ / tertiary;
(C) be a hydrogen donor for a hydrogen bond/ secondary;
(D) be a hydrogen donor for a hydrogen bond/ tertiary.

       A       10. BPG forms                                                with the                            part of hemoglobin.
(A) ionic interactions/ globin (protein); (B) ionic interactions/ heme;
(C) hydrogen bonds/ heme; (D) hydrophobic interactions/ globin.

11-14. Use diagrams X-Z and this sequence for Z (N → C): S A L E I F C K T V L T Y M S A.

      A        11. Diagram X represents a/n              β-sheet with sequence                for the top strand.
(A) parallel, E → H; (B) parallel, H → E;
(C) antiparallel, E → H; (D) antiparallel, H → E.  
(E → H means E is nearer the amine terminus.)

      C        12. Diagram Z represents an α-helix, with H and K showing the positions of                      , and I showing the position of                 . (A) side chains/ α-carbonyl;
(B) α-amines/ side chain; (C) α-amines/ α-carbonyl; (D) all are side chains.

      B        13. The residues labeled E, F, and G on the α-helix are most likely to be
(A) A, L, and E; (B) E, K, and T; (C) F, C, and K; (D) L, V, and S.
Remember to check the sequence at the top.

      A        14. An interaction that helps to stabilize this α-helix is a/n (A) ionic interaction between glutamate and lysine; (B) hydrophobic interaction between valine and alanine; (C) ionic interaction between histidine and lysine; (D) hydrophobic interaction between phenylalanine and tryptophan.

15-16. Use this amino acid, which has an α-COOH pK_a= 1.88, and α-NH₃⁺ pK_a= 9.60, and a side chain pK_a= 3.65. The amino acid is shown in different protonated forms.

      D        15. Write the one-letter abbreviation for this amino acid in the blank.

      D        16. Given its pK_a's, which form is most common at pH 3.3?

      C        17. The intrinsic favorability of a reaction can be given by ΔG°′ or by K_eq . Which of these reactions is most favorable?
(A) ΔG°′ = + 3.5 kJ/mol; (B) K_eq = 0.004; (C) ΔG°′ = – 25 kJ/mol; (D) K_eq = 0.99.

      B        18. Compared to leucine, polyleucine (with four or more residues) would be
                 soluble in water, with a pK_afor the α-amine that is more                .
(A) more/ acidic; (B) less/ acidic; (C) more/ alkaline; (D) less/ alkaline.

Comment: As the α-amine moves farther from the α-COO^-, it becomes easier for the H⁺ to leave at a higher [H⁺], which is the same as a more acidic pH.

      C        19. Other amino acids can also be combined into homopolymers (polyglutamate, polylysine, etc.). Which homopolymer would be the most like leucine?
(A) polyglycine; (B) polyaspartate; (C) polyvaline; (D) polytyrosine.

      B        20. Think of graphing θ for hemoglobin as a function of pO₂ in the absence of BPG. Adding BPG shifts the curve                                 , and lowering the pH causes                     .
(A) to the left/ even more of a shift to the left;
(B) to the right/ even more of a shift to the right; (C) to the right/ no effect;
(D) to the right/ a shift back to the left.

      C        21. One difference between Ribonuclease A (RNase A) and most other proteins is that RNase A (A) is denatured by urea; (B) is denatured by sulfuric acid;
(C) can regain its native conformation after being denatured; (D) all of the above.

      A        22. Cells avoid being damaged by ribonuclease A by (A) synthesizing inhibitors that bind it tightly; (B) causing it to denature; (C) breaking its disulfide bonds;
(D) packaging it into vesicles that are extruded from the cell.

      D        23. The y axis of a Hill plot is         , and the slope is n, which shows the              .
(A) log pO₂/ number of binding sites; (B) log θ / number of subunits;
(C) θ / % of binding sites occupied;
(D) log (θ/(1-θ)/ O₂-affinity of the binding sites.

      C        24. What is the effect of increasing [H⁺] on hemoglobin?
(A) The α-chain COO^- is protonated and forms a hydrogen bond with O₂ ;
(B) The β-chain NH₂ is protonated and is attracted to BPG;
(C) A histidine side chain is protonated and is attracted to an Asp^- side chain;
(D) A histidine side chain is protonated and repels Fe²⁺.

      B        25. The T conformation of hemoglobin is stabilized by        , so that O₂ is                   .
(A) CO₂ / bound; (B) CO₂ / released; (C) O₂ / bound; (D) H⁺ / bound.

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