Terms in bold are defined in the glossary.
1. Properties of the Peptide Bond In x-ray studies of crystalline peptides, Linus Pauling and Robert Corey found that the bond in the peptide link is intermediate in length (1.32 Å) between a typical single bond (1.49 Å) and a double bond (1.27 Å). They also found that the peptide bond is planar (all four atoms attached to the group are located in the same plane) and that the two α-carbon atoms attached to the are always trans to each other (on opposite sides of the peptide bond).
2. Structural and Functional Relationships in Fibrous Proteins William Astbury discovered that the x-ray diffraction pattern of wool shows a repeating structural unit spaced about 5.2 Å along the length of the wool fiber. When he steamed and stretched the wool, the x-ray pattern showed a new repeating structural unit at a spacing of 7.0 Å. Steaming and stretching the wool and then letting it shrink gave an x-ray pattern consistent with the original spacing of about 5.2 Å. Although these observations provided important clues to the molecular structure of wool, Astbury was unable to interpret them at the time.
3. Rate of Synthesis of Hair α-Keratin Hair grows at a rate of 15 to 20 cm/yr. All this growth is concentrated at the base of the hair fiber, where α-keratin filaments are synthesized inside living epidermal cells and assembled into ropelike structures (see Fig. 4-10). The fundamental structural element of α-keratin is the α helix, which has 3.6 amino acid residues per turn and a rise of 5.4 Å per turn (see Fig. 4-3a). Assuming that the biosynthesis of α-helical keratin chains is the rate-limiting factor in the growth of hair, calculate the rate at which peptide bonds of α-keratin chains must be synthesized (peptide bonds per second) to account for the observed yearly growth of hair.
4. Effect of pH on the Conformation of α-Helical Secondary Structures Specific rotation is a measure of a solution’s capacity to rotate circularly polarized light. The unfolding of the α helix of a polypeptide to a randomly coiled conformation is accompanied by a large decrease in a property called specific rotation. Polyglutamate, a polypeptide made up of only l-Glu residues, is an α helix at pH 3. When researchers raise the pH to 7, there is a large decrease in the specific rotation of the solution. Similarly, polylysine (l-Lys residues) is an α helix at pH 10, but when researchers lower the pH to 7, the specific rotation also decreases, as shown in the graph.
Explain the effect of the pH changes on the conformations of poly(Glu) and poly(Lys). Why does the transition occur over such a narrow range of pH?
5. Disulfide Bonds Determine the Properties of Many Proteins Some natural proteins are rich in disulfide bonds, and their mechanical properties, such as tensile strength, viscosity, and hardness, correlate with the degree of disulfide bonding.
6. Dihedral Angles Consider the series of torsion angles, ϕ and ψ, that might be taken up by the peptide backbone. Which of these closely correspond to ϕ and ψ for an idealized collagen triple helix? Refer to Figure 4-8 as a guide.
7. Amino Acid Sequence and Protein Structure Our growing understanding of how proteins fold allows researchers to make predictions about protein structure based on primary amino acid sequence data. Consider this amino acid sequence.
8. Amino Acid Contributions to Protein Folding Like ribonuclease A, lysozyme from T4 phage is a model enzyme for understanding the energetics and pathways of protein folding. Unlike ribonuclease A, however, T4 lysozyme does not contain any disulfide bonds. A number of studies have quantified the thermodynamic contributions that individual amino acid residues and their interactions make to T4 lysozyme folding.
9. Bacteriorhodopsin in Purple Membrane Proteins Under the proper environmental conditions, the salt-loving archaeon Halobacterium halobium synthesizes a membrane protein known as bacteriorhodopsin, which is purple because it contains retinal (see Fig. 10-20). Molecules of this protein aggregate into “purple patches” in the cell membrane. Bacteriorhodopsin acts as a light-activated proton pump that provides energy for cell functions. X-ray analysis of this protein reveals that it consists of seven parallel α-helical segments, each of which traverses the bacterial cell membrane (thickness 45 Å). Calculate the minimum number of amino acid residues necessary for one segment of α helix to traverse the membrane completely. Estimate the fraction of the bacteriorhodopsin protein that is involved in membrane-spanning helices. (Use an average amino acid residue weight of 110.)
10. Conservation of Protein Structure Margaret Oakley Dayhoff originated the idea of protein superfamilies after noticing that proteins with diverse amino acid sequences can have similar tertiary structures. Why can protein structure be more highly conserved than individual amino acid sequences?
11. Interpreting Ramachandran Plots Examine the two proteins labeled (a) and (b) below. Which of the two Ramachandran plots, labeled (c) and (d) at right, is more likely to be derived from which protein? Why? [Data from (a) PDB ID 1GWY, J. M. Mancheno et al., Structure 11:1319, 2003; (b) PDB ID 1A6M, J. Vojtechovsky et al., Biophys. J. 77:2153, 1999.]
12. Number of Polypeptide Chains in a Multisubunit Protein A researcher treated a sample (660 mg) of an oligomeric protein of with an excess of 1-fluoro-2,4-dinitrobenzene (Sanger’s reagent) under slightly alkaline conditions until the chemical reaction was complete. He then completely hydrolyzed the peptide bonds of the protein by heating it with concentrated HCl. The hydrolysate was found to contain 5.5 mg of the compound shown.
2,4-Dinitrophenyl derivatives of the α-amino groups of other amino acids could not be found.
13. Amyloid Fibers in Disease Several small aromatic molecules, such as phenol red (used as a nontoxic drug model), have been shown to inhibit the formation of amyloid in laboratory model systems. A goal of the research on these small aromatic compounds is to find a drug that efficiently inhibits the formation of amyloid in the brain in people with incipient Alzheimer disease.
14. Protein-Folding Therapies The Food and Drug Administration recently approved the drug lumacaftor for the treatment of cystic fibrosis in patients with the F508ΔCFTR mutation. This mutation is a genetically encoded deletion of amino acid F508 from the protein. About of cystic fibrosis patients have this mutation, and lumacaftor is one of the first drugs that functions as a pharmacological chaperone to correct a defect in the protein-folding process. However, lumacaftor is not always effective in treating patients who have other CFTR mutations that result in misfolding. Why is lumacaftor able to correct the misfolding of some mutant CFTR proteins and not others?
15. Structural Biology Methods Which structural biology method (CD, x-ray crystallography, NMR, or cryo-EM) is best suited to each task?
16. Using the PDB The Protein Data Bank (PDB) contains more than 150,000 different three-dimensional biomolecular structures obtained by x-ray crystallography, NMR, and cryo-EM. Each protein structure deposited into the database is given a PDB ID. Several PDB IDs represent proteins whose structures resemble letters from the Roman alphabet. Find each protein structure in the PDB and view the three-dimensional structure using JSmol, PYMOL, or a similar structure viewer.
PDB IDs: 2QYC, 2BNH, 2Q5R, 1XU9, 3H7X, 1OU5, 2WCD
17. Protein Modeling Online A group of patients with Crohn disease (an inflammatory bowel disease) underwent biopsies of their intestinal mucosa in an attempt to identify the causative agent. Researchers identified a protein that was present at higher levels in patients with Crohn disease than in patients with an unrelated inflammatory bowel disease or in unaffected controls. The protein was isolated, and the following partial amino acid sequence was obtained (reads left to right):
EAELCPDRCI |
HSFQNLGIQC |
VKKRDLEQAI |
SQRIQTNNNP |
FQVPIEEQRG |
DYDLNAVRLC |
FQVTVRDPSG |
RPLRLPPVLP |
HPIFDNRAPN |
TAELKICRVN |
RNSGSCLGGD |
EIFLLCDKVQ |
KEDIEVYFTG |
PGWEARGSFS |
QADVHRQVAI |
VFRTPPYADP |
SLQAPVRVSM |
QLRRPSDREL |
SEPMEFQYLP |
DTDDRHRIEE |
KRKRTYETFK |
SIMKKSPFSG |
PTDPRPPPRR |
IAVPSRSSAS |
VPKPAPQPYP |
18. Mirror-Image Proteins As noted in Chapter 3, “The amino acid residues in protein molecules are almost all l stereoisomers.” It is not clear whether this selectivity is necessary for proper protein function or is an accident of evolution. To explore this question, Milton and colleagues (1992) published a study of an enzyme made entirely of d stereoisomers. The enzyme they chose was HIV protease, a proteolytic enzyme made by HIV that converts inactive viral preproteins to their active forms.
Previously, Wlodawer and coworkers (1989) had reported the complete chemical synthesis of HIV protease from l-amino acids (the l-enzyme), using the process shown in Figure 3-30. Normal HIV protease contains two Cys residues, at positions 67 and 95. Because chemical synthesis of proteins containing Cys is technically difficult, Wlodawer and colleagues substituted the synthetic amino acid l-α-amino-n-butyric acid (Aba) for the two Cys residues in the protein. In the authors’ words, this was done to “reduce synthetic difficulties associated with Cys deprotection and ease product handling.”
Wlodawer and coworkers denatured the newly synthesized protein by dissolving it in 6 m guanidine HCl and then allowed it to fold slowly by dialyzing away the guanidine against a neutral buffer (10% glycerol, 25mm , pH 7).
In a more recent study, Milton and coworkers synthesized HIV protease from d-amino acids, using the same protocol as the earlier study (Wlodawer et al.). Formally, there are three possibilities for the folding of the d-protease: it would be (1) the same shape as the l-protease, (2) the mirror image of the l-protease, or (3) something else, possibly inactive.
In fact, the d-protease was active: it cleaved a particular synthetic substrate and was inhibited by specific inhibitors. To examine the structure of the d- and l-enzymes, Milton and coworkers tested both forms for activity with d and l forms of a chiral peptide substrate and for inhibition by d and l forms of a chiral peptide-analog inhibitor. Both forms were also tested for inhibition by the achiral inhibitor Evans blue. The findings are given in the table.
Inhibition | |||||
---|---|---|---|---|---|
HIV protease | Substrate hydrolysis | Peptide inhibitor | Evans blue (achiral) | ||
d-substrate | l-substrate | d-inhibitor | l-inhibitor | ||
l-protease |
− |
+ |
− |
+ |
+ |
d-protease |
+ |
− |
+ |
− |
+ |