• Biochemistry 655 Fall 2007

CASE STUDIES IN STRUCTURAL MOLECULAR BIOLOGY

M,W,F 10:00-10:50 Rm 305 Faculty Laboratory and Office Building

C.W. Carter, Jr.

323A Octy Ellen Jones

966-3263

Office Hours by Appointment

Textbooks: (* = required)

               *Fersht, A., (1999 Structure and Mechanism in Protein Science (F)

                                                 Freeman, San Francisco

               Petsko, G. and Ringe, D. Protein Structure and Function (P&R)

                                                 Sinauer, Sunderland, MA

               Hecht, Sidney M. (1998) Bioorganic Chemistry: Peptides and Proteins

Oxford University Press, New York (Chapter 5: Protein Structure

                                                 sumOctizes the material taught in Biochemistry 134 as of 1998 and

                                                 evolved from the class notes.)

               Finkelstein, A. V. and Ptitsyn, O. B. Protein Physics, Academic Press, London, UK

                                                 This book is a treasure! It travels far beyond the heights we hope to scale,

                                                 but is essential for anyone seriously interested in penetrating to deeper levels.

Description of Goals

All case study references, occasional supplementary notes and Kinemages are available from the syllabus as hyperlinks. :

http://xtal.med.unc.edu/CARTER/carter_WWW/Bioch_655/KIN

Kinemages require the public domain program, MAGE, available at:

http://kinemage.biochem.duke.edu

General references helpful over a range of case studies are given at the beginning of some of the units.

Study questions are to help you think about and discuss the Case studies. They are not necessarily suggestive of what might be on examinations.

Powerpoint and Keynote files: KNT_PPT

Feedback is always welcome in any form, and is especially important at the end of the course.

Jump to Case Study 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37 38 39 40 41 42 43

Please note that because of my schedule later in the semester, some class periods will be changed in some way; either by proceeding without me and reporting to me afterward or by meeting at another time suitable to everyone.

Date	Case Study Topic	Objective/Study Questions	Reference/Notes
22-Aug 0	Ligand-linked conformational change: Riboswitches riboswitch_movie.ppt	This ppt presentation introduces one of the guiding themes of this course in an entirely novel context 1. What is a riboswitch? 2. Where are they found? 3. What is the relationship between the concentration of metabolite and the operational state of the riboswitch?	(Mandal and Breaker, 2004)
UNIT I	OVERTURE: INTRODUCTION TO STRUCTURAL STUDIES (4 sessions)	This preliminary overview builds a basis for understanding ideas central to image formation, working from one, through two, to three dimensions. Examples illustrate how material will be developed from other case studies. Tropomyosin illlustrates the “grail” of understanding how sequence determines function; Tubulin provides a preview of an NTPase; Bacteriorhodopsin illustrates the use of weak acids and bases in proteins to move protons against a gradient and how the spontaneous reverse process is used for chemical work.	P&R Chapt. 5 Branden and Tooze, Ch 18
24-Aug Cs1	One-Dimensional Fourier and Transforms and PriOcty Sequence Analysis Problem I Due Kinemage files: c3Alpha.kin, Tropo.kin, demo2_4a.kin Ozgun and Gurkan	Calculate a one-dimensional Fourier transform; understand its use in identifying patterns 1. What two objects are related by Fourier Transformation in this case study? 2. Why do clusters of carboxylate side chains repeat roughly every 19.6 residues, rather than every 21 residues in Tropomyosin? 3. Why would one expect them to repeat every 21 residues?	[P&R] § 2-5. CWCJr; Chapt 5, P164-169 (McLachlan, 1976; Stewart, 1975)
27-Aug Cs2	Two and three-dimensional Fourier transforms: Kinemage files: c13Membr.kin, tubulin.kin, 1Ftsz.kin Steven and Erin	The crystallographic phase problem and its solution via Fourier transformation 1. How do amplitudes and phases combine to form an image? 2. Where do the amplitudes and phases come from in electron crystallography? 3. To what substructure does the cofactor bind in Tubulin?	(Nogales et al., 1995; Unwin, 1984) Notes: Electroncryst.htm
29-Aug Cs3	Making and using proton gradients Kinemage file: BR.kin; ATP_synth.kin Lauren and Jun	Hydrogen ion is a central “ligand” involved in Bioenergetics. Its movements are carefully managed in energy storage and utilization. 1. What is common to the generation of a proton gradient across a membrane and to its conversion to mechanical energy? 2. What role do conformation changes play in the two processes?	[F], PP 317-321; (Fillingame, 1999; Gennis & Ebrey, 1999; Luecke et al., 1999; Stock et al., 1999) Notes: BR_F1_notes.hrm
UNIT II	LINDERSTRØM-LANG'S HIERARCHY (9 Sessions)	Proteins are organized at four levels, arranged hierarchically. We have already anticipated the study of the hierarchy in the Tropomyosin case study in UNIT I. Here, we investigate more deeply questions associated with the development of higher levels from the sequence.	[P&R] Chapt. 1 (Cordes et al., 1996; Dill, 1990; Dill & Fersht, 1996) Notes: Sequence_space.note
31-Aug Cs4	Primary, Secondary, Tertiary, and Quaternary Structure; Backbone dihedral angles and hydrogen bonding Kinemage files: c1Basic.kin, c2Motifs.kin; c7DNAbd.kin, c8TrnscF.kin; EX9nNovo.kin Aaron and Zier Note: Carter out of town unless we can meet at 8:00-9:00	Local determinants of folding Distinguish between local and global interactions. Are any local interactions likely to be unusually influential? What kinds of data inform us about the role of local interactions – how can we draw useful conclusions?	[P&R] § 1-8 CWCJr Chapt 5, p 161-201; Fersht, p 1-34. (Muñoz & Serrano, 1994) (Butterfoss & Hermans, 2003) Notes: Local_dets.notes
5-Sept Cs5	The Folding Landscape Ozgün and Stephen	The Levinthall Paradox; compensation of energy and entropy in the overall free energy Is it possible to sustain a “native” conformation without limiting the number of accessible states? What kinds of interactions can reduce the number of states? How might protein folding recapitulate protein evolution?	[F] p 575-602 (Dinner et al., 2000; Onuchic et al., 1997) ** Focus on pp 545-560 of Onuchic paper!! Notes: Landscape.notes
7-Sept Cs6	Hydrophobic bonding, likelihood potentials, and tertiary templates Kinemage files: tessell.kin Gürkan and Erin	Bioinformatic approaches to the study of tertiary interactions 1. How well does the “puzzle-pieces” model describe packing? (See also Butterfoss, 2003.) 2. What advantages accrue when considering multiple contacts?	CWCJr Chapt 5, P186-188 (Carter et al., 2001; Ponder & Richards, 1987; Tropsha et al., 1996) Notes: Tert_templ.notes
10-Sept Cs7	No Class; Carter away
12-Sept Cs8	Folds and genomics Problem Set II due Kinemage files: c3Alpha.kin,c5Beta.kin, c4Al_Bet.kin Lauren and Aaron NB: need to reschedule; 8:00 PM?	Bioinformatics: Fold Classification and Covariation. The right descriptors and the right ruler. How is cluster analysis a reciprocal process? How many dimensions are involved in the classification scheme of Chou? How well does the database support a simple classification scheme?	CWCJr Chapt 5, P. 184-198; [F] p 25-33 (Hou, Jun et al., 2005; Bahar et al., 1997; Michie et al., 1996) Notes: Classify_folds_families.notes
14-Sept Cs9	F,m-analysis- I Kinemage files: Barnase_phi_values.kin Erin and Ozgün	Mutational analysis of protein folding: Kinetics and transition states How does mutation localize effects in space? How does Fersht localize effects in time? What is the reOctkable conclusion from combining localization in time and space?	[F] Chapt 17-18 (Matouschek et al., 1990; Matouschek et al., 1989) Notes: Fersht_I.notes
17-Sept Cs10	F,m-analysis- II Lauren and Gürkan	A discrete pathway; a limited number of parallel paths Why is this question important? How does Fersht answer it? What connections are there between this work and the concept of the “folding landscape”?	[F] Chapt 18-19 (Daggett et al., 1996; Dalby et al., 1998; Fersht et al., 1994; Otzen et al., 1994) Notes: Bronsted.notes
19-Sept Cs11	Quaternary structure Kinemage files: nucleosome.kin PROBLEM III Jun and Aaron	Hemocyanin, Nucleosome illustrate the functionality of broken symmetry What are the relative advantages and disadvantages of high symmetry in macromolecular interactions? What compromises are necessary to resolve these conflicts?	[P&R] § 1-19 – 1-21 CWCjr, p 199-210 (Lamy et al., 1981; Luger et al., 1997; Xie et al., 1996) Notes: Hemocyan.notes
21-Sept Cs12	Conformational change I Kinemage files: hemagglut.kin Steven and Zier	Hemagglutinin pH transition illustrates conformational regulation and its use in infection. On what basis was the haemagluttinin transition predicted? What does the low pH structure tell us about protein stability?	[P&R] Chapt 1-22, 3-5 [F] Chapt 10. CWCjr, p 215-218; (Bullough et al., 1994; Carr & Kim, 1993)
24-Sept Cs13	Conformational change II Kinemage files: T7_RNA_polym.kin Lauren and Ozgün	Transition from initation to elongation involves extensive refolding of viral RNA polymerase. 1. What is “processivity”? 2. How does the polymerase rearrange after initiation in order to ensure a processive completion of translation?	(Yin & Steitz, 2002)
UNIT III	BINDING INTERACTIONS IN WATER (7 Sessions)	Protein stability and binding affinity both arise from differences between interactions in water and those that obtain inside proteins. Key questions include cooperativity and how binding and conformational free energies are interconverted.	[F] Chapt 11 [P&R] § 2-0 -– 2-4.
26-Sept Cs14	Modularity: Intracellular signaling-I Kinemage files: poly_pro.kin, collagen.kin, c14recep.kin Gürkan and Jun	Modules; polyproline II binding is a widespread motif How does the incorporation of proline into a repeating polypeptide induce the “polyproline II” structure? What aspects of the PP II structure lend themselves to protein-protein interactions?	(Huang et al., 2000; Verdecia et al., 2000; Zarrinpar & Lim, 2000)
28-Sept Cs15	Modularity: Intracellular signalling-II Kinemage files: Steven and Aaron	SH2 and Phosphoserine-binding motifs What interactions distinguish between phospho and dephospho peptides? How does phosphorylation affect the activation of cSrc?	(Waksman et al., 1993; Young et al., 2001)
1-Oct Cs16	Hemoglobin-I Kinemage files: Hemoglobin.kin PROBLEM SET IV due Erin and Lauren	Quaternary constraints create the low-affinity states of oligomeric proteins. Hemoglobin is the “hydrogen atom” of structural biology. Why is it surprising that the weakest affinity binding site is filled first? Why are the lowest affinity binding sites the hardest to create? How does conformation change create low-affinity binding sites? What does positive cooperativity entail? How does it work?	[P&R] § 1-19—1-22 (Perutz et al., 1998)
3-Oct Cs17	Hemoglobin-II Aaron and Erin	Internal Coupling. A quintessential Rube Goldberg mechanism for intramolecular signalling. What changes in Iron chemistry are communicated through the Hb tertiary structure, and how are they communicated? How are the magnetic properties of Iron used to demonstrate the linkage between ligand binding and conformation change?	(Dickerson & Geis, 1984; Perutz, 1978)
5-Oct Cs18	Tryptophanyl-tRNA synthetase Kinemage files: ilyin.kin Lauren and Steven	Linking binding to conformation change. This fundamental concept permeates all of biology. How many binding sites does TrpRS have? Which of these might be linked to conformation changes? How is the linkage effected?	[F] 44-50; 324-347 (Ilyin et al., 2000)
8-Oct Cs19	NLS Recognition Kinemage files: Karyo.kin Jun and Ozgün	Peptide binding and the intracellular circulation of proteins. What is unusual about the tertiary structure of a-karyopherin? Which amino acids are most important for peptide recognition, and what distinctive roles do they play?	(Conti et al., 1998)
10-Oct Cs21	ATP-dependent binding-III MHC complex formation-I Kinemage files: c12immun.kin Gürkan and Aaron	MHC complex formation and antigen presentation is representative of a series of interactions that require low specificity together with a slow off rate. How is this compromise achieved in the MHC systems? What must be true of the systems that package peptides for presentation on the surface of cells?	(Garboczi et al., 1996; Garcia et al., 1996; Jardetzky et al., 1996; Watts, 1997)
12-Oct	Midterm Exam (Due 12/10) (Due 15 October)	The midterm examination will cover all previously covered material. It will be take-home and consist of a variety of quantitative and/or essay questions.
UNIT IV	CATALYSIS: STRUCTURAL AND MECHANISTIC ENZYMOLOGY (5 sessions)	Catalysis emerges when a protein “learns” to differentiate between the structure of a molecule and its structure in the transition state for a chemical transformation of the same compound. We know a substantial amount about how enzymes work from a variety of historic and contemporary approaches.	[F] Chapt 2-4, 7,8, 10 [P&R] § 2-6 – 2-16
15-Oct Cs22	Equilibrium models; transition state stabilization Kinemage files: c15serpr.kin; cda_animate_2.kin PROBLEM V due Jun and Steven	Differential binding energy and catalysis 1 What is the difference between a transition state and a transition state analog? What does it mean that enzymes are considered to be in equilibrium with transition states in solution?	(Carlow et al., 1998; Wolfenden, 1991) (Robertus, 1972)
17-Oct Cs23	Directed mutagenesis Aaron and Ozgün	Perturbation methods facilitate the localization of binding energy. What fundamental problem is associated with using directed mutagenesis to study enzyme mechanisms> How are these obstacles overcome in practice?	(Carter, 1988; Fersht, 1985)
19-Oct Cs24	No Class; Fall Break!!