Chapter Review

KEY TERMS

Terms in bold are defined in the glossary.

Problems

A four-part figure, a, b, c, and d, shows protein structures as ribbon structures in parts a and b and two Ramachandran plots in parts c and d.
BIOCHEMISTRY ONLINE
  • 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

    1. For each protein, identify its quaternary structure and describe the protomer structure as all α, all β, α/β, or α + β.
    2. What letter does each protein structure most closely resemble?
    3. What word(s) can you spell using these protein structures?
  • 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

    1. You can identify this protein using a protein database such as UniProt (www.uniprot.org). On the home page, click on the link for a “BLAST” search. On the BLAST page, enter about 30 residues from the protein sequence in the appropriate search field and submit it for analysis. What does this analysis tell you about the identity of the protein?
    2. Try using different portions of the amino acid sequence. Do you always get the same result?
    3. A variety of websites provide information about the three-dimensional structure of proteins. Find information about the protein’s secondary, tertiary, and quaternary structures using database sites such as the Protein Data Bank (PDB; www.rcsb.org) or Structural Classification of Proteins (SCOP2; http://scop2.mrc-lmb.cam.ac.uk).
    4. In the course of your online searches, what did you learn about the cellular function of the protein?
DATA ANALYSIS PROBLEM
  • 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.”

    1. The structure of Aba is shown below. Why was this a suitable substitution for a Cys residue? Under what circumstances would it not be suitable?
      A structure shows L-alpha-Amino-n-butyric acid. The molecule has a central carbon bonded to N H 3 plus below, bonded to C H 2 further bonded to C H 3 on the right, bonded to C above that is double bonded to O and bonded to O minus, and bonded to H on the left.

      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 NaH2PO4/Na2HPO4NaH Subscript 2 Baseline PO Subscript 4 slash Na Subscript 2 Baseline HPO Subscript 4, pH 7).

    2. There are many reasons to predict that a protein synthesized, denatured, and folded in this manner would not be active. Give three such reasons.
    3. Interestingly, the resulting l-protease was active. What does this finding tell you about the role of disulfide bonds in the native HIV protease molecule?

      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.

    4. For each possibility, decide whether or not it is a likely outcome, and defend your position.

      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

      +

      +

      +

    5. Which of the three models proposed is supported by these data? Explain your reasoning.
    6. Why does Evans blue inhibit both forms of the protease?
    7. Would you expect chymotrypsin to digest the d-protease? Explain your reasoning.
    8. Would you expect total synthesis from d-amino acids followed by renaturation to yield active enzyme for any enzyme? Explain your reasoning.

References