1.  Matters of stability. Proteins are quite stable. The lifetime of a peptide bond in aqueous solution is nearly 1000 years. However, the free energy of hydrolysis of proteins is negative and quite large. How can you account for the stability of the peptide bond in light of the fact that hydrolysis releases considerable energy?

2.  Name those components. Examine the segment of a protein shown below. ✓ 2



(a) What three amino acids are present?

(b) Of the three, which is nearest the N-terminal amino acid?

(c) Identify the peptide bonds.

(d) Identify the α-carbon atoms.

3.  Who’s charged? Draw the structure of the dipeptide Gly-His. What is the charge on the peptide at pH 4.0? At pH 7.5?

4.  First abbreviate, then charge. Examine the following peptide, and answer parts a through c.

Thr-Glu-Pro-Ile-Val-Ala-Pro-Met-Glu-Tyr-Gly-Lys

(a) Write the sequence using one-letter abbreviations.

(b) Estimate the net charge at pH 7.

(c) Estimate the net charge at pH 12.

5.  Neighborhood peer pressure? Table 3.1 gives the typical pK_a values for ionizable groups in proteins. However, more than 500 pK_a values have been determined for individual groups in folded proteins. Account for this discrepancy.

6.  Prohibitions. Why is rotation about the peptide bond prohibited, and what are the consequences of the lack of rotation? ✓ 2

7.  Don’t they make a lovely pair? Match the terms with the descriptions. ✓ 2

8.  Helix length. Calculate the axial length of an α helix that is 120 amino acids long. How long would the polypeptide be if it were fully extended?

9.  Alphabet soup. How many different polypeptides of 50 amino acids in length can be made from the 20 common amino acids? ✓ 2

10.  Sweet tooth, but calorie conscious. Aspartame (Nutra-Sweet), an artificial sweetener, is a dipeptide composed of Asp-Phe in which the carboxyl terminus is modified by the attachment of a methyl group. Draw the structure of aspartame at pH 7.

11.  Vertebrate proteins? What is meant by the term polypeptide backbone?

12.  Not a sidecar. Define the term side chain in the context of amino acid or protein structure.

13.  One from many. Differentiate between amino acid composition and amino acid sequence. ✓ 2

14.  Knowledge is good. List some of the benefits of knowing the primary structure of a protein. ✓ 2

15.  Compare and contrast. List some of the differences between an α helix and a β strand. ✓ 2

16.  Degrees of complication. What are the levels of protein structure? Describe the type of bonds characteristic of each level. ✓ 2

17.  Two by two. Match the terms with the descriptions. ✓ 2

18.  Who goes first? Would you expect Pro–X peptide bonds to tend to have cis conformations like those of X–Pro bonds? Why or why not? ✓ 2

19.  Contrasting isomers. Poly-l-leucine in an organic solvent such as dioxane is a helical, whereas poly-l-isoleucine is not. Why do these amino acids with the same number and kinds of atoms have different helix-forming tendencies? ✓ 2

20.  Active again. A mutation that changes an alanine residue in the interior of a protein into valine is found to lead to a loss of activity. However, activity is regained when a second mutation at a different position changes an isoleucine residue into glycine. How might this second mutation lead to a restoration of activity? ✓ 3

21.  Exposure matters. Many of the loops on the proteins that have been described are composed of hydrophilic amino acids. Why? ✓ 2 ✓ 3

22.  Goodbye native state. Hello chaos. How would each of the following treatments contribute to protein denaturation? ✓ 3

(a) Heat

(b) Addition of the hydrophobic detergents

(c) Large changes in pH

23.  Often irreplaceable. Glycine is a highly conserved amino acid residue in the evolution of proteins. Why? ✓ 3

24.  Potential partners. Identify the groups in a protein that can form hydrogen bonds or electrostatic bonds with an ar-ginine side chain at pH 7.

25.  Permanent waves. The shape of hair is determined in part by the pattern of disulfide bonds in keratin, its major protein. How can curls be induced?

26.  Location is everything 1. Most proteins have hydrophilic exteriors and hydrophobic interiors. Would you expect this structure to apply to proteins embedded in the hydrophobic interior of a membrane? Explain. ✓ 3

27.  Location is everything 2. Proteins that span biological membranes often contain α helices. Given that the insides of membranes are highly hydrophobic, predict what type of amino acids will be in such α helix. Why is an α helix particularly suitable for existence in the hydrophobic environment of the interior of a membrane? ✓ 3

28.  Greasy patches. The a and β subunits of hemoglobin bear a remarkable structural similarity to myoglobin. However, in the subunits of hemoglobin, residues that are hydrophilic in myoglobin are hydrophobic. Why? ✓ 3

29.  Maybe size does matter. Osteogenesis imperfecta displays a wide range of symptoms, from mild to severe. On the basis of your knowledge of amino acid and collagen structure, propose a biochemical basis for the variety of symptoms.

30.  Scrambled ribonuclease. When performing his experiments on protein refolding, Christian Anfinsen obtained a quite different result when reduced ribonuclease was reoxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1% of the enzymatic activity of the native protein. Why were the outcomes so different when reduced ribonuclease was reoxidized in the presence and absence of urea? ✓ 3

31.  A little help. Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of β-mercaptoethanol were added to an aqueous solution of the protein. Explain these results. ✓ 3

32.  Shuffle test. An enzyme called protein disulfide isomerase (PDI) catalyzes disulfide–sulfhydryl exchange reactions. PDI rapidly converts inactive scrambled ribonuclease into enzymatically active ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence of insulin and its three-dimensional structure? ✓ 3

33.  Stretching a target. A protease is an enzyme that catalyzes the hydrolysis of the peptide bonds of target proteins. How might a protease bind a target protein so that its main chain becomes fully extended in the vicinity of the vulnerable peptide bond?

34.  V = 4/3 πr³. Consider two proteins, one having a molecular weight of 10,000 and the other having a molecular weight of 100,000. Both are globular and of similar spherical shape. How will the ratio of hydrophilic to hydrophobic amino acids differ between the two proteins?

35.  Shape and dimension. Tropomyosin, a 70-kDa muscle protein, is a two-stranded α-helical coiled coil. Estimate the length of the molecule.

36.  Concentration is crucial. The concentration of RNase that Anfinsen used when performing his renaturation experiments was approximately 1 mg ml⁻¹. Inside a cell, the protein concentration is estimated to be more than 100 mg ml⁻¹. Predict the outcome of Anfinsen’s experiments had he used a 100 mg ml⁻¹ RNase.