A Research Style Biochemistry Lab: Collaborating on

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

2. HistoricalBiochemistry: 1828 synthesis of urea 1833 isolation of amylase 1896 fermentation using yeast extracts 1903 general acceptance of the term “biochemistry” 1962 ACS publication of Biochemistry 1998 ASC biochemistry requirement Interdisciplinary aspects: Analytical Cell biology Organic Microbiology Biochemistry Molecular biology Inorganic Physical Genetics

3. Curricular goals: • Explore fundamental biochemistry techniques • Teach experimental design and data interpretation • Expose chemistry students to interdisciplinary pedagogy • Make connections between molecular structure and function • Reinforce concepts from lecture

4. Research focus: • Hypothesis driven • Continuity • Open-ended Biochemistry Research • Explorations into the functional or structural properties • of isolated biological molecules under • controlled conditions

5. Design implementation: Activated methyl cycle and methionine biosynthesis • Defects in methionine pathway • elevated homocysteine • increased ROS • arteriosclerosis Cystathionine-b-Lyase (CBL) (E. coli) Steegborn, C., et al., Kinetics and inhibition of recombinant human cystathionine gamma-lyase – Toward the rational control of transsulfuration. Journal of Biological Chemistry, 1999. 274(18): p. 12675-12684.

6. Cystathionine-b-Lyase (CBL) Uren, J. R. (1987). "Cystathionine Beta-Lyase From Escherichia-Coli." Methods In Enzymology143: 483-486.

7. Design implementation: • Colorimetric assay for product formation • Commercially available substrates • Complex reaction mechanism • Crystal structure

8. Cystathionine-b-Lyase (CBL) pyridoxal 5’-phosphate • Complex reaction mechanism • Crystal structure 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.

9. The Process 1) Each student group generates a hypothesis • analysis of reaction mechanism and enzyme active site “I think the hydroxyl group on tyrosine 111 stabilizes substrate binding”

10. The Process: 2) Each group designs a mutant to test their hypothesis

11. 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

12. Advantages of silent mutation • Use of bioinformatics software (EMBOSS) • Review genetic code (protein DNA) • Predict outcome of restriction digests (NEB cutter 2.0) • Avoid the “black box” of DNA sequencing • Students empowered to “order” DNA oligomer • and restriction enzyme

13. 1 2 3 4 5 6 The Process: 3) DNA isolation and analysis • Standard kit isolation • Compare restriction digests of wild type and mutant DNA • *silent mutation adds a restriction site Bfa I digest of plasmid DNA Lane 1: 1kb DNA ladder Lane 2 – 5: non-mutant CBL plasmid DNA Lane 6: mutant CBL plasmid DNA (Y111F) Larissa Nordstrom, Chrissie Chow, Rachel Dyer (2006)

14. The Process: 4) Protein expression, isolation and analysis Affinity chromatography Bradford assay SDS-PAGE

15. The Process: 5) Enzyme kinetics (functional analysis) • Three substrates • Wild-type and mutant enzyme • Different pH buffers Experimental Design: measure d[P] dt [S] = ??? Km = [S] at ½ Vmax (Km values from literature) [E] = ??? [S] >> [E] determined through trial and error

16. Results: 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

17. The Process: 6) Each group shares results in a final presentation or report • Revisit hypothesis “I think the hydroxyl group on tyrosine 111 stabilizes substrate binding” • Evaluate calculations 70 fold change 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

18. Lessons learned Units, units, units!!!! Never underestimate the difficulty of a simple calculation Perspective – how much is reasonable? when is a change significant? Always provide a standard template for reporting results There is a bridge across the river

19. Is this publishable?

20. 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 exactly what we did in Chem 321 lab” -from a student who worked as a research tech at Mayo after graduation. Overall sense of empowerment and ownership of their mutants

21. Acknowledgements Carleton College, Department of Chemistry 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