MACROMOLECULAR STRUCTURE AND FUNCTION, BIOSC 1810 Date: 12/2016 FALL TERM, 2016

INSTRUCTOR Dr. Paula Grabowski Email: [email protected] Office: A502B Langley Hall inside lab (Fifth floor of Langley Hall across from cold room) TEACHING ASSISTANTS

OFFICE HOURS Each week ample office hours will be provided. See “OFFICE HOURS” link on CourseWeb. CLASS MEETINGS Lectures: MWF 1:00 – 1:50 PM, 169 Crawford Hall Recitations: Thurs 12:00-12:50 & 1:00-1:50 PM, 241 Crawford Hall COURSE DESCRIPTION Biochemistry is a systematic way of thinking about, and experimentally probing, life’s processes at the scale of atoms and molecules. Chemical concepts, quantitative methods, and experimental reasoning will be the framework in which we explore macromolecular structure, function. There are four modules: (1) protein structure, function and analysis, (2) enzyme kinetics and inhibition, (3) RNA enzymes and their mechanisms, and (4) membrane transporters and ion channels. In addition to textbook readings, you will have exposure to the scientific literature throughout the course for real world illustrations of experimental tools and approaches. Pre-requisites: 2-semesters of Foundations of Biology, General Chemistry, and Organic Chemistry COURSE OBJECTIVES 1. You will be able to use the language and reasoning of biochemistry to explain the following core concepts: a. Levels of protein structure, motifs, conformational changes, allosteric movements, folding/unfolding; Chromophore structure/function b. Receptor-ligand affinity: stereochemistry; equilibrium dissociation constants c. Enzyme mechanisms and inhibition at the active site; substrate and transition state analogs, suicide inhibitors, feedback inhibition; RNA enzymes/ribozymes d. Membrane chemistry & architecture; active and passive transport 2. You will be able to apply the following experimental approaches, techniques, and tools to design, and critically assess, experiments aimed at answering specific questions: a. Protein purification and analytical separation methods, including column chromatography, isoelectric focusing, and ultracentrifugation b. Protein sequencing and analysis, including MALDI and ESI mass spectrometry, Sanger’s reagent; proteases; bioinformatics resources c. X-ray diffraction as an approach to atomic level structure/function; use of transition state analogs to pinpoint or modulate the active site d. FRET as used in high-throughput enzyme assays; FRET sensors e. Michaelis-Menten kinetics as an approach to understanding enzyme mechanisms, including the steady state approximation, kcat, Km, and kcat/Km parameters; pH dependence of enzymatic reactions f. Ribozyme engineering through SELEX 3. You will build self-confidence as you gain practice reading challenging, peer-reviewed scientific publications! MASTERY IS THE SECRET Master the material in each module of the course by solving the problem-based worksheets provided TO A STRONG to you. Attend all lectures and recitations. Keep pace with the reading and take home assignments, PERFORMANCE IN THIS and do them carefully. Find a regular place to study for this course that will minimize distractions and COURSE help you to focus on learning. Build regular study time for this course into your schedule. COURSEWEB AND Course materials including lecture notes will be posted under Course Documents. You will be MASTERY FOLDER responsible for checking regularly for updates to reading assignments, and Take home assignments. The “Mastery folder”, which is a valuable resource for you, will contain worksheets with exam-style questions for you to master as you learn the course material and prepare yourself for the exams. REQUIRED READING • Principles of Biochemistry, by Lehninger, Nelson and Cox, 6th Edition, 2013. Copies of the MATERIALS textbook are on reserve in Langley Library (2 hour reserve). It will be most helpful to use the 6th edition! • Posted papers will be made available to you on CourseWeb as supplementary reading. SCIENTIFIC CALCULATOR You will need a standard scientific calculator for this course. It will be used for the three interim exams and the final exam. Bring the calculator with you to recitations.

1 GRADING • EXAMS • Exams are weighted at 80 points each. (240 points) • Exams will be taken closed book with a standard scientific calculator permitted. No electronic devices are permitted. • Exams are cumulative. • Excepting typos, questions of clarification are NOT permitted during exams. • If you miss an exam, you will need to have a compelling excuse in the form of written documentation, such as a signed letter from a physician or the equivalent.

• FINAL EXAM • The Final Exam will be given at the date and time scheduled by the university Registrar. (120 points) • The Final Exam is required. It will cover material after Exam 3, and it will be cumulative. • The Final Exam will not be returned to you; you can examine it by appointment. • If you have three or more final exams in one day, you may request an alternate final exam time. Please see the Final Exam Conflict Accommodation Procedure (http://www.registrar.pitt.edu/assets/pdf/final_procedure.pdf).

• TAKE HOME Written assignments will be completed outside of the classroom. Two, 15-point assignments will be ASSIGNMENTS (120 scheduled for each of the four modules of the course. Instructions and materials will be posted on points) Courseweb. For maximum points, follow all directions and do careful, original work. Please note: excepting a valid excuse, the consequence of turning in a late assignment is that the assignment will not be graded. So, make sure your work is turned in on time.

• Final grades Your grade will be determined by the sum of the following components (480 points in total): Exams 240 points (50%), Final exam 120 points (25%), and Assignments 120 points (25%). Prior to assigning final grades the class average will be adjusted to a C+/B-, if necessary. Final grades will be assigned based on the following scale: percentages in the 90's = A range; 80's = B range; 70's = C range; 60's = D range; 50 or below = F. RECITATION The purpose of recitation is to solve problems related to the course material, and to answer questions about the posted papers. The questions will emphasize quantitative issues, fundamental concepts, experimental procedures, results, and hypotheses. LEADERSHIP TEAM This course is like a journey up a steep mountain. We are your guides for the journey. You will need to exercise reasonable self-discipline to keep pace. ACADEMIC INTEGRITY Students in this course will be expected to comply with the University of Pittsburgh's Policy on Academic Integrity. Any student suspected of violating this obligation for any reason during the semester will be required to participate in the procedural process, initiated at the instructor level, as outlined in the University Guidelines on Academic Integrity. This may include, but is not limited to, the confiscation of the examination of any individual suspected of violating University Policy. Furthermore, no student may bring any unauthorized materials to an exam, including dictionaries and programmable calculators. EMAIL POLICY • Questions about course policies, absences, grades, and letters of recommendation should be directed to the instructor. You will receive a response within 24-hours. DISABILITY SERVICES If you have a disability for which you are or may be requesting an accommodation, you are encouraged to contact both your instructor and Disability Resources and Services (DRS), 140 William Pitt Union, (412) 648-7890, [email protected], (412) 228-5347 for P3 ASL users, as early as possible in the term. DRS will verify your disability and determine reasonable accommodations for this course. COPYRIGHT NOTICE Course materials may be protected by copyright. United States copyright law, 17 USC section 101, et seq., in addition to University policy and procedures, prohibit unauthorized duplication or retransmission of course materials. See Library of Congress Copyright Office and the University Copyright Policy. STATEMENT ON To ensure the free and open discussion of ideas, students may not record classroom lectures, CLASSROOM RECORDING discussion and/or activities without the advance written permission of the instructor, and any such recording properly approved in advance can be used solely for the student’s own private use.

2 SCHEDULE OF TOPICS & ASSIGNMENTS – for full details see LECTURES & READING on our CourseWeb site

Broad topics Specific breakdown Week 1 M 08/29 Introduction • Biochemistry: risks and rewards posted paper by Ringe & Pesko • The story of James B. Sumner and Urease; active sites and induced fit; Review Chapter 1 Some of biochemistry’s modern rewards: pharmaceuticals

W Amino acids and peptides Amino acids: Acid base chemistry & Henderson-Hasselbalch; ionization Chapter 3 sections 3.1, 3.2, 3.3; state of His (Ch3, prob 4) Chapter 2 section 2.3

Th Recitation pH and drug absorption (Ch2, prob15); aspirin’s mechanism-of-action with respect to inactivation of COX; graphical analysis

F Working with proteins I Column chromatography for separation and purification; workflow and Chapter 3, 3.3, 3.4 validation; proteases as biochemical tools; net electric charge of peptides; isoelectric focusing Week 2 M 09/05 Labor Day Holiday

W Working with proteins II Sanger’s approach to sequencing Insulin; mass spectrometry Chapter 3, 3.4 & prob 23 approaches, isoelectric focusing

Th Recitation Enzyme purification tables for validation, sequence logos, use of proteases, two-dimensional gels

F Secondary and tertiary motifs Secondary and tertiary motifs: non-covalent interactions; alpha helices; Chapter 4, 4.1, 4.2 alpha helices in tertiary folds; EF-hand motif; Shimadzu link for MS-MS (http://www.shimadzu.com/an/lifescience/maldi/maldi7090_5.html) Week 3 M 09/12 Protein denaturation & folding continued from Fri, Overview of protein structure: secondary and Chapter 4, 4.3, 4.3 tertiary motifs, non-covalent interactions, protein denaturation and folding, role of water

W Christian Anfinsen’s hypothesis Scrambled Ribonuclease experiment; free energy funnels; protein Chapter 4, 4.4; Posted paper by chaperones Anfinsen

Th Recitation Chapter 4 probs 1, 4, 12, 14; review scrambled ribonuclease expt

F Vitamin B12: Dorothy Crowfoot Hodgkin’s development of x-ray crystallography to Chapter 17, BOX 17-2 study cholesterol, Vitamin B12, penicillin, AND Insulin! Fourier Posted paper by Gruber, transforms and role of heavy atoms Introduction and 1.1 (structure) Week 4 M 09/19 Green fluorescent Protein I Osamu Shimumura’s efforts to purify Green Fluorescent Protein from Posted paper by Shimumura tons of jellyfish! Purification from light organs, column Chapter 12: Box 12-3 chromatography, sea water, Aequorin

W Green fluorescent Protein II b-can tertiary fold; Greek key motifs; chromophore assembly and Posted paper by Ormo; Chapter structure; procedural steps of the Ormo paper 12, Box 12-3

Th Recitation GFP

F Modern applications of GFP FRET sensors; mass spectrometry posted paper by Heim Week 5 M 09/26 Protein-ligand interactions Monoclonal antibodies, Immunoglobulin fold, Fab fragments, induced Chapter 5, 5.2; Ch 5 probs 1, 5 fit; protein-ligand affinity, dissociation constants; antibody-drug

3 Posted paper by Hess conjugates

W EXAM 1, 80 points will cover material from weeks 1-4

Th Recitation cancelled

F Cooperativity: Hemoglobin Cooperative binding, Hill coefficient, allosteric effectors, Hemoglobin Chapter 5, 5.1 & probs 6 and 8 variants Week 6 M 10/03 Enzyme catalysis How enzymes work, Rate enhancements by entropy reduction; Chapter 6, 6.1, 6.2 and probs 4,7 transition states

Enzyme kinetics Steady state assumption, initial rates, total enzyme, kcat, Km, and W Chapter 6, 6.3 and probs 8, 21 kcat/Km; applications of the Michaelis Menten equation

Th Recitation Herceptin video and questions; quantitative analysis of receptor-ligand binding; Pathological variants of Hemoglobin

F Kinetics and inhibition I Continued Chapter 6 and probs 11, 12, 13 Week 7 M 10/10 Kinetics and inhibition II pH activity profiles; ribonuclease active site structure and function; Chapter 6, 6.3 and probs 21, 23 Posted paper by Thompson

W Enzyme engineering Enzyme engineering of Lactate dehydrogenase Wilks posted paper

Th Recitation

F Chymotrypsin mechanism II Chymotrypsin enzyme structure and active site; evidence for an acyl- Hedstrom posted paper enzyme intermediate; pre-steady state kinetics; sections I and II of Hedstrom Week 8 M 10/17 Fall Break Class moved to Tuesday 10/18

T Chymotrypsin mechanism II Roles of Ser195, His57, Asp102 in the Chymotrypsin mechanism; Hedstrom posted paper inhibition by isopropylfluorophosphate; pH activity profiles

W Allosteric enzyme regulation I ATCase I: pyrimidine biosynthesis; catalytic and regulatory subunits; Chapter 6, 6.5 role of Zn2+; Schachman’s differential centrifugation expt

Th Recitation

F Allosteric enzyme regulation II ATCase II: structure in the T and R states; interactions with bisubstrate Posted paper by Lipscomb analog, PALA

Week 9 M 10/24 Nucleotides and nucleic acids I Nucleotide and nucleic acid structure; base modifications; helix-coil Chapter 8, 8.1, 8.2, 8.3 transitions; alkaline hydrolysis of RNA; R-loop mapping

W EXAM 2, 80 points will cover material from week 5-8

Th Recitation Cancelled

F RNA structure rules Free energy minimization; base stacking interactions; loop closure Tinoco and Bustemante penalties; sequence co-variation Week M 10/31 Self-cleaving ribozymes Introduction to Hammerhead structure & function; rolling circle 10 genome replication; introduction to SELEX

W SELEX approaches for ribozyme Discuss figure facts on Szostak paper, including design principles, 4 discovery methods, and results Posted paper by Szostak

Th Recitation Melting curves; R loop mapping; experimental design; figure 3 of Szostak figure facts

F Computational approaches for Comparative genomics, covariance models, homology searches based ribozyme discovery on conservation of secondary structure; discovery of high speed Perreault posted paper hammerhead ribozymes in all branches of life including humans Week M 11/7 Self-splicing Group 1 introns I Secondary structure and mechanism of the self-splicing Group I intron 11 Guo posted paper; chapter 26, of Tetrahymena; role of guanosine cofactor 26.2 for background W Self-splicing Group I introns II Internal guide sequence; tetraloop and tetraloop receptor, base triple Guo posted paper continued; sandwich; metal ion binding sites Butcher posted paper (tertiary structure motifs)

Th Recitation

F Crawling out of the RNA World Role of contemporary RNA enzymes; ribozyme engineering Posted paper by Cech Week M 11/14 Lipids and membranes Structural lipids in membranes; lipids as signals, cofactors, and 12 Chapter 10, 10.1 10.2, 10.3 pigments; steroid hormones, fat-soluble vitamins

W EXAM 3, 80 points Will cover material from weeks 9-11

Th Recitation cancelled

F Solute transport through Integral membrane proteins and their interactions with lipids; membranes hydropathy plots; transporter types; catalysis of solute transport Chapter 11, 11.1, 11.2, 11.3; Chapter 11 prob 13 (lipid melting temperatures, prob 15 (membrane Ch11 probs 13, 15, 22 permeability), prob 22 (use of the helical wheel diagram) Week M 11/21 Na+K+ ATPase Pump I Active transport; Na+K+ ATPase pump, discovery, biological functions, 14 Posted paper by Kuhlbrandt; Ch sequence conservation 11.3 for background

W Thanksgiving recess, Safe travels! Wednesday 11/23 – Sunday 11/27 Week M 11/28 Na+K+ ATPase Pump II Free energy of transport calculations; protein architecture; mechanism 15 Posted paper by Kuhlbrandt of action; Post-Alber’s cycle; inhibition by cardiotonic steroids

W Aquaporin I The problem of water transport; Grotthuss mechanism; discovery of Posted paper by Preston aquaporins by Peter Agre (also see Gateways by Miller)

Th Recitation

F Aquaporin II Mechanism of water transport; molecular dynamics simulations as an Posted paper by Sui approach to understand mechanism WEEK M 12/05 K+ Channels I Introduction; K+ channel signature sequence function and 16 Chapter 11, 11.3; Posted paper conservation; Roderick MacKinnon’s vision to “see” the channel at high by Heginbotham resolution

W K+ Channels II MacKinnon’s failed attempts to crystalize the KcsA channel and the Posted paper by Zhou breakthrough procedure; specificity and mechanism of K+ conduction; 5 the selectivity filter; high speed conduction by “knock-on”

Th Recitation Worksheet on Zhou and Preston experimental procedures; review alpha helix

F K+ Channels II channel gating mechanisms; voltage sensing; channelopathies

FINAL FRI DEC 16 FROM 2:00-3:50 PM Where? 169 Crawford Hall

6