115:412/508       PROTEINS AND ENZYMES      spring 2001

Text: This course covers a mixture of practical approaches to proteins - measurement, purification, assay of enzyme actrivity - characterization of proteins and their functions, and mechanism of enzymes.  No single book adequately covers all these topics.  I have ordered at the Douglass-Cook bookstore Protein Analysis and Purification Benchtop Techniques, I. Rosenberg, Birkhäuser, 1996, Protein Purificat­ion, R.K. Scopes, Springer-Verlag, 3rd ed., 1993, and Structure and Mechanism in Protein Science, A. Fersht, W.H. Freeman & Co., 1999, which is actually the 3rd edition of a previous text, Enzyme Structure and Mechanism .  The first covers not only protein purification (the next nine lec­tures or so) but methods of characteri­zation of proteins, including their post-translational modifications, a topic of in­creasing importance (four lectures) and a number of other modern techniques with which I am not personally familiar.  The Scopes book is the best for protein purification, and the bookstore also has it for Dr. Ward's course Biochemical Separations, which gives a fuller coverage of protein purification than this course.  The Fersht book is good on mech­anism of enzyme action (last six lectures) and gives some coverage of other topics.   This edition has extensive cover­age of the mechanism of tyrosyl-tRNA synthetase, which I lectured on previously, based on Fersht's papers on it.  The Rosenberg book intends to be a practical guide to design of experiments as well as alternative explanations of what I lec­ture about (you will find it helpful to read the suggested reading before the lecture).  You probably do not, for this course, have to get both Rosenberg and Scopes, but if you are engaged in protein purification and characterization you probably should have them both.  I do not spend any time on primary, secondary and tertiary structure of proteins, or on methods of determining them (sequencing, X-ray crystallography, nuclear magnetic resonance); however, I usu­ally ask Dr. Kahn to give one lecture on protein structure modeling in the computer.  Two useful books on structure are Proteins Structures and Molecular Properties, T.E. Creighton, W.H. Freeman & Co., 1984, 2nd ed., 1992, and Introduc­tion to Protein Structure, C. Brandén and J. Tooze, Garland Publ., 1991.   I am placing these books on reserve at the Chang Library (Cook campus), and some others :

      Fundamentals of Enzymology, N.C. Price & L. Stevens, Oxford , 1982 (P&S below)
      The Enzyme Molecule, W. Ferdinand, John Wiley & Sons, 1976
      The Enzymes, P.D. Boyer ed., Academic Press, 3rd ed.,  vols. 1 & 2, 1970, vol. 11, 1975, vol. 19, l990
      Proteins and Enzymes, J.E. Bell & E.T. Bell, Prentice-Hall, 1988
In addition, a number of references are given to Annual Review of Biochemistry (available in the Library of Science & Medicine), and to specific papers (which often I have used in preparing the lectures).

Examinations: papers from the biochemical literature, which you are to read (at home) and answer questions on.  The three examinations will comprise 10, 20 and 30% of the grade, and the course paper 40%.

Course paper: an original research proposal, 5 to 10 pages long, which identifies an important question about an enzyme or other protein, plans one or more experiments designed to answer the question, and indicates what sort of results might be expected and how they would answer the question.  This should not be your thesis proposal, though it may be a related question in the general area of your thesis project.  Early discussion of ideas with me is strongly suggested (in particular to ensure that it is a protein proposal, not a molecular biology proposal; in 1996 I had to ask that several papers be re-done).  It should not be just a review*, or an enzyme purification, or an application of a routine procedure, but should show imaginative thinking and tight reasoning.  It is generally better to choose a relatively small, well-defined question, to which the experimental approach is clear, rather than the larger, more open-ended question one might pursue in actual research.  The paper is due at the last regular meeting of the class (April 30).  *Undergraduates have the option of doing a review paper rather than a research proposal.

Lecture topics, with suggested reading (approximate, I often fall behind, the visiting speakers may be rearranged if they have other commitments, and I might bring in new material, or skip a lecture at the end if I am behind schedule):

Jan. 17 General introduction.  Amino acids, primary, secondary and tertiary structure: Rosenberg pp. 5-18, Fersht pp. 8-24.  Natural sources vs. recombinant (Scopes pp. vii-viii, 270-1; Rosenberg pp. 325-7).  Purify first, then think.  Specific activity.  Nomenclature. P&S 1-13, Ferdinand pp. 1-13.

Jan. 22 Determination of protein concentration: Rosenberg pp. 110-7, Scopes pp. 44-50, 349-350, Creighton pp. 25-28, Stoschek, Meth. in Enzymol. 182:50-68 (1990).  Assays of activity: general considerations.

Jan. 24 Assays of activity: general considerations, methods of measurement, continuous vs. stop-time. P&S pp. 116-8, Scopes pp. 50-62, Fersht pp. 191-8, Ferdinand pp. 36-40, Allison & Purich, Meth. in Enzymol. 63:3-22 (1979).  Buffers: Rosenberg pp. 374-380, Scopes pp. 324-333, 351-2.

Jan. 29 Important types of assay.  Coupled assays: Scopes pp. 63-70, Rudolph et al., Methods in Enzymol. 63: 22-42 (1979)  Cycled assays.  Immunological assays.  Assay without knowing your protein: Marvel & Kammen, Anal. Biochem. 181:336-340 (1989), Rosenberg pp. 19-24  (327-334 for in vitro translation).

Jan. 31 Purification of proteins.  The purification table.  Basic procedures: Scopes pp. 1-21.  Extraction: Scopes pp. 22-43, Rosenberg pp. 99-109.  Activation, inactivation: Rosenberg pp. 117-9, Scopes pp. 317-324, P&S pp. 15-19, 27-41, Ferdinand pp. 82-4, 115-7.   Recombinant proteins: Schein, Bio/Technology 7:1141-8 (1989), Rosenberg pp. 335-350, Scopes pp. 270-277.  Membrane proteins: Rosenberg pp. 135-152.

Feb. 5 Precipitation methods:  Rosenberg pp. 119-120, 124-133, Scopes pp. 71-101.  Ethanol precipitation: van Oss, J. Prot. Chem. 8:661-8 (1989).  Partitioning: Scopes pp. 264-7, Baskir et al., Biotech. Bioeng. 34: 541-558 (1989), Huddleston et al., J. Chromatogr. A 668:3-11 (1994), Biotech Bioeng. 44:626-35 (1994).

Feb. 7  Chromatography:  general principles: Scopes pp. 102-121, 139-145,  P&S pp. 19-23, Rosenberg pp. 265-7.  Batch adsorption: Scopes pp. 121-6, Rosenberg pp. 288-9.  Ion exchange etc.: Rosenberg pp. 283-296, Scopes pp. 135-8, 146-175, .

Feb. 12 Hydrophobic chromatography, immobilized metal ion chromatography, etc.: Rosenberg pp. 296-300, Scopes pp. 175-186.  Gel filtration: Rosenberg pp. 267-279, Scopes pp. 238-250.  High performance liquid chromatography: Rosenberg pp. 279-283, 300-303, Scopes pp. 126-132.

Feb. 14 Affinity chromatography, immunoadsorption, dye-ligand chromatography: Rosenberg pp. 34-41, 303-324, Scopes pp. 180-237, P&S pp. 24-7, Meth. in Enzymol. vol. 34, Affinity Chromatography (books by Lowe & Dean, Scouten).  Affinity partitioning: Luong & Nguyen, Bio/Technology 8:306-7 (1990); metal ion affinity partitioning: Birkenmeier et al., J. Chromatogr. 539:267-277 (1991).  Take-home Exam handed out.

Feb. 19 Preparative electrophoresis, electrofocusing, chromatofocusing: Scopes pp. 250-263, Rosenberg pp. 292-4.  Analyt­ical electrophoresis, criteria of purity, mol. wt. determination: Rosenberg pp. 55-98, P&S pp. 27-9, 44-50, Scopes pp. 293-309, Ferdinand pp. 47-50, 272-5.  Transfer of proteins to membranes, detection: Rosenberg pp. 153-180.  Small scale, large scale: Scopes pp. 283-292. 

Feb. 21 Review of basic enzyme kinetics: P&S pp. 118-126, Ferdinand pp. 21-36, Fersht pp. 103-114, Creighton pp. 405-409.  Statistics: Wilkinson, Biochem. J. 80:324-332 (1961), Cleland, Adv. in Enzymol. 27:1 (1968), Fersht pp. 209-214.

Feb. 26    Acyl-enzyme intermediates: P&S pp. 163-4, Fersht pp. 216-231. Pre-steady state kinetics: Fersht pp. 132-155.   Active site titration: Fersht pp. 155-8, P&S pp. 126-136, Creighton p. 416. Exam due.

Feb. 28 Kinetics with two or more substrates: P&S pp. 136-142, 161-2, Fersht pp. 119-122, Creighton pp. 409-410, Cleland, The Enzymes  vol. 2 pp. 1-18, Biochim. Biophys. Acta  67:104-137 (1963), Dalziel, Acta Chem. Scand.  11:1704-1733 (1957), The Enzymes  vol. 11 pp. 1-15.

Mar. 5 Effects of inhibitors and pH on enzyme kinetics: Fersht pp. 112-7, 169-189, Ferdinand pp. 138-164.  Product inhibition: P&S pp. 142-3, Cleland, The Enzymes  vol. 2 pp. 18-43.  Substrate inhibition.

Mar. 7 Binding: Fersht pp. 202-9, Creighton pp. 335-347.  Allosteric enzymes, mnemonic enzymes: Fersht pp. 289-308, Creighton pp. 380-396, P&S pp. 208-233.  Organized enzyme systems: Rosenberg pp. 41-54, P&S pp. 265-307, Fersht pp. 34-8; Srere, Ann. Rev. Biochem. 56:89-124 (1987).  It is unlikely that I will be able to cover both these topics - I have rarely been able to get to even one of them.

                              VACATION

Mar. 19 Chemical modification of proteins: Rosenberg pp. 19-31, Fersht pp. 273-7, Creighton pp. 6-24, Cohen, The Enzymes  vol. 1 pp. 147-211, Means & Feeney, Chemical Modification of Proteins, Feeney, Int. J. Pepti. Prot. Res. 29: 145-161 (1987).

Mar. 21 Active-site-directed modification: P&S pp. 170-172, Fersht pp. 277-280, Creighton pp. 414-415, Shaw, The Enzymes  vol. 1 pp. 91-146, Colman, Ann. Rev. Biochem. 52:67-91 (1983).  Suicide substrates: Fersht pp. 280-6, Walsh, Ann. Rev. Biochem. 53:493-535 (1984).  Slow, tight-binding inhibition: Fersht pp. 286-7.  Photoaffinity labeling: Fleming, Tetrahedron 51:12479-12520 (1995), Brunner, Ann. Rev. Biochem. 62:483-514 (1993).  Take-home exam handed out.

Mar. 26    Site-specific mutagenesis: what can the protein biochemist learn?  Fersht pp. 420-7 (methods pp. 413-5), Leatherbarrow & Fersht, Protein Eng. 1:7-16 (1986), Ackers & Smith, Ann. Rev. Biochem. 54: 597-629 (1985), Knowles, Science 236:1252-1258 (1987).  Folding and stabilization: Matthews & Hurle, Bio­Essays 6:254-257 (1987), Brandén & Tooze pp. 257-260.  Linear free energy relationships: Fersht pp. 442-4, Fersht et al., Nature 322:284-6 (1986), Kirsch, Protein Eng. 1:148-150 (1987), Straub & Karplus, Protein Eng. 3:673-5 (1990).  Redesign: Fersht pp. 450-4.  Incorpora­tion of unnatural amino acids: Noren et al., Science 244:182-8 (1989), Ibba & Hennecke, Bio/Technology 12:678-682 (1994)  Selenocysteine: Böck et al., Mol. Microbiol. 5:515-520 (1991), Stadt­man, Ann. Rev. Biochem. 65:83-100 (1996)

Mar. 28    Post-translational modification: Rosenberg pp. 207-8, 257-60, Uy & Wold, Science  198:890-6 (1977), Wold, Ann. Rev. Biochem.  50: 783-804 (1981), Creighton pp. 70-87, Methods Enzymol.vols. 106, 107.  Protein disulfide forma­tion/ iso­merization: Noiva, Protein Express. Purif. 5:1-13 (1994).  Conversion of l- to d- amino acids: Kreil, Science 266:996-7 (1994), Heck et al, Science 266:1065-8 (1994), Keil, Ann. Rev. Biochem 66:337-346 (1997).  Protein splicing: Fersht p. 26, Cooper & Stevens, BioEs­says 15:667-674 (1993), Perler et al., Nucl. Acids Res. 22:1125-7 (1994), Xu et al., EMBO J. 13:5517-22 (1994), 15:5146-5153 (1996).  Take-home exam due.

Apr.  2     Fatty acylation: Rosenberg  pp. 245-7, Towler et al., Ann. Rev. Biochem. 57:69-99 (1988), Gordon et al., J. Biol. Chem. 266:8647-50 (1991), Casey, Current Opinion in Cell Biology 6:219-225 (1994).  Prenylation and methylation: Rosenberg pp. 247-251, Clarke, Ann. Rev. Biochem. 61:355-386 (1992), Omer & Gibbs, Mol. Microbiol. 11:219-225 (1994), Zhang & Casey, Ann. Rev. Biochem. 65:241-269 (1996).  Glypiation: Rosenberg pp. 251-7.  Glycosylation:  Rosenberg pp. 208-224.

Apr. 4 Phosphorylation/dephosphorylation (kinases/phosphatases): Dr. Michael Lawton.  Rosenberg pp. 224-244

Apr. 9 Mechanisms of catalysis: P&S pp. 152-161, Fersht pp. 54-96, Creighton pp. 397-405, Bruice, The Enzymes  vol. 2 pp. 217-229.  Kinetic isotope effects: Fersht pp. 96-99.  Intermediates: Fersht pp. 216-243, Creighton pp. 414-419.

Apr. 11     Rate-limiting steps: Fersht pp. 158-167, Dunford & Hasinoff, J. Inorg. Biochem. 28: 263-269 (1986), Ray , Biochemistry  22:4625-4637 (1983), Yasigawa, Biochem. J. 263:985-8 (1989).  Forces between mole­cules, binding energy: Fersht pp. 324-347.  Use of binding energy: Fersht pp. 349-375.  Transition states, transition state analog design: Schramm, Ann. Rev. Biochem. 67:693-720 (1998).

Apr. 16 Mechanism of serine proteases: Fersht pp.  26-30, 40-43, 218-230, 473-482, Brandén & Tooze pp. 231-245. 

Apr. 18 Mechanism of tyrosyl-tRNA synthetase: Fersht pp. 422-450, 235-242; Schimmel, Ann. Rev. Biochem. 56:125-158 (1987), Carter, Ann. Rev. Biochem. 62:715-748 (1993), Brandén & Tooze pp. 51-3, 260-263; Wells & Fersht, Biochemistry  25:1881-6 (1986), Fersht, Biochemistry  26:8031-7 (1987), First & Fersht, Biochem­istry 32:13644, 13651, 13658 (1993).

Apr. 23 Alcohol dehydrogenases: Fersht pp. 249-250, 458-465, P&S pp. 197-205, Brandén & Tooze pp. 141-151,
Brandén et al. The Enzymes vol. 11 pp. 104-171, Maret & Makinen, J. Biol. Chem. 266:20636-44 (1991)

Apr. 25 Protein modeling in the computer: Dr. Peter Kahn.  This lecture will be in Lipman 202.  Take-home exam handed out.

Apr. 30 Catalytic antibodies: Benkovic, Ann. Rev. Biochem. 61:29-54 (1991), Tramontano et al., Science 234: 1566-1570 (1986), Pollack et al., Science 234:1570-1572 (1986), Blackburn et al., Biochem. J. 262:381-390 (1989),J. Am. Chem. Soc. 111:9261 (1989), Powell & Hansen, Protein Eng. 3:69-75 (1989), Campbell et al., Proc. Nat. Acad. Sci. 90:8663-7 (1993).  COURSE PAPER DUE.

May 4      Take-home exam due.