A Research Style Biochemistry Lab: Collaborating on the Integration of Research and Teaching at Two Institutions

1. A Research Style Biochemistry Lab: Collaborating on the Integration of Research and Teaching at Two Institutions Joseph Chihade Department of Chemistry Carleton College Gregory W. Muth Department of Chemistry St. Olaf College

2. The origins of this project…

3. Curricular Goals: • Expose students to fundamental techniques • Reinforce concepts from lecture • Teach experimental design and data interpretation A “Research-Style” lab: • Continuity throughout the term • Hypothesis driven • Open-ended

4. Some institutional differences:

5. What we needed: A gene for an enzyme: A structure:

6. The opportunity: Genome sequencing Availability of structural data From: National Center for Biotechnology Information From: San Diego Supercomputer Center

7. Modular design: Structural Biology: Students use protein structure and enzymatic reaction mechanism to form hypotheses, design mutations. 1. Molecular biology: Students use site directed mutagenesis to create mutants and analyze restriction patterns to check success. 2. Biochemistry: Students purify mutant protein and assess effect of mutation by comparing kinetics with mutant and wildtype enzymes. 3.

8. Or… Last year’s work Biochemistry: Students purify mutant protein and assess effect of mutation by comparing kinetics with mutant and wildtype enzymes. 3. Structural Biology: Students use protein structure and enzymatic reaction mechanism to form hypotheses, design mutations. 1. Molecular biology: Students use site directed mutagenesis to create mutants and analysis of restriction patterns to check success. 2. Next year’s work

9. Our choice: Cystathionine-b-Lyase (CBL) Clausen, T., R. Huber, et al. (1996). "Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 angstrom." Journal of Molecular Biology262(2): 202-224.

10. Biological/Medical Relevance: Activated methyl cycle and methionine biosynthesis Defects in the methionine pathway Elevated homocysteine ➔ increased ROS ➔ arteriosclerosis

11. Part 1: Make a hypothesis. On what basis do students formulate a hypothesis? • Analysis of the enzyme’s structure. • Knowledge about the chemical mechanism. • A database of results from previous students’ work at both colleges. The value of a naïve hypothesis. • We strive to go with students’ choices. • Sometimes “daring” mutations are the most interesting ones.

12. An example: “I think the hydroxyl group on tyrosine 111 stabilizes substrate binding”

14. Part 2: Make a mutant. Mutagenesis with additional silent mutation CBL DNA acc aac acc gcc tat gaa ccg agt cag gat T N T A Y111 E P S Q D CBL protein sequence mutant CBL T N T A F111 E P S Q D mutant DNA acc aac acc gcc ttt gaa cct agt cag gat Second change introduces or removes a restriction site, no change in protein sequence – silent mutant

15. WT • Compare restriction digests of wild type and mutant DNA • *silent mutation adds a restriction site Bfa I digest of plasmid DNA Potential mutants Larissa Nordstrom, Chrissie Chow, Rachel Dyer (2006)

16. Part 3: Characterize the mutant. Affinity chromatography to purify protein Assessment of purity Quantitation

17. Enzyme kinetics: Group 1 Group 2 CBL CBL Km = 94 mM kcat = 82 sec-1 Km = 54 mM kcat = 58 sec-1 Y111F CBL S339A CBL Km = 28 mM kcat = 0.81 sec-1 Km = 30 mM kcat = 0.038 sec-1

18. Putting it together. • Revisit hypothesis “I think the hydroxyl group on tyrosine 111 stabilizes substrate binding” • Evaluate calculations 70 fold reduction in kcat Minimal change in Km • Conclude The placement of Y111 within the active site (distant from PLP) along with the kinetic data suggest that the Y111 hydroxyl helps position the substrate in an optimal orientation for the chemical reaction

19. Lessons we’ve learned Units are still a stumbling block. Never underestimate the difficulty of a simple calculation Initial “quick and dirty” assay can help students gain perspective. Tension between standardization and open ended writing in reporting results. There is a bridge across the river.

20. The value of collaboration Technical help is just a phone call away! Pedagogical conversation is too! Building up a database of knowledge more quickly than we otherwise would. Students can build on each other’s hypotheses. There is a bridge across the river.

21. So, can you publish this data? We think so! • Need replication and validation of results Students who are interested continue work as independent project Both models allow replicates to be run in subsequent years.

22. Is this really like research? Student Perceptions: “Experimental Biochem. Lab does apply to the real world!!!!” - Hayley Ross ‘07, while doing summer research at the University of Pittsburgh “I do pretty much exactly what we did in Chem 321 lab.” - Paul Letendre ‘05, on his work as a research technician at Mayo after graduation. Overall sense of empowerment and ownership of their mutants

23. Acknowledgements Carleton College, Department of Chemistry Spring 04-05 Alice Agyiri Andrew Berry Stephan Chapman Stephanie Contag Mark Ericson Katie Freeman Sara Ganaha Vickie Gunderson David Jackson Emily Johnson Paul Letendre Robbie McDermott Thaibinh Nguyen Claire Richardson Kartik Sampath Sinele Tsabedze Kate Waller Dorissa Zemirah Spring 05-06 Erin Addison Disan Davis Thayne Dickey Jon Dozier Michael Draper Randy Foss Dani Hargreaves Admire Kuchena Won Lee Alex Marston Cam Noland Shayna Simmons Matt Topeff Ellen Valkevich Will VanStory Lauren Wagner Chris Ward Maya Warren Pete Watson Amanda Winters St. Olaf College, Faculty and Students Fall 05-06 Brennan Decker Kiyomi Goto Mike Kuprian Colin Reily Hayley Ross Chris Torstenson Spring 05-06 Nisar Baig Chrissie Chow Rachel Dyer Christine Gille Liz Johnson Matt Majerus Brandon Moriarty Larissa Nordstrom Fall 06-07 Andrew Bodger Colette Cave Tyler Drake Sultan Mirzoyev James Morrison Pat Nelson Paul Nichol Katherine Oyster Ryan Ritzer