1. PJK 20081 Using HX-MS to Determine Protein Structure of AhpC 2, a Peroxiredoxin Piper J. Klemm Faculty Advisor: Claudia S. Maier, PhD HHMI Fellowship, OSU August 20, 2008

2. PJK 20082 Hypothesis Mass spectrometers (MS) can be used as tools to elucidate protein structure through proton exchange with their solvent. This structure can be used for the determination of protein functions and dynamics.

3. PJK 20083 Hydrogen Exchange with Solvent Deuterium solvent exchanges with structural protons without change in structure (A) Protein unfolded in exchange process (B) Wales & Engen 2006.

4. PJK 20084 Alpha Helices Hydrogen bonding in alpha helices Limits proton-solvent exchange http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg

5. PJK 20085 Plenty of surface area for solvent interaction http://cnx.org/content/m11614/latest /beta_sheet_cartoon.JPG Beta Sheet

6. PJK 20086 Background HX-MS to analyze protein structure www.hxms.com/hxms.htm

7. PJK 20087 Secondary Structure Background http://www.rcsb.org/pdb/explore/remediatedSequence.do?st ructureId=1YF0&params.chainEntityStrategyStr=all&forceP ageForChain=E#chainC

8. PJK 20088 Protein Map Goal Protein Map for PPARγ LBD. Each block has six distinct time points color coordinated with deuterium level (bottom right). Hamuro, Yoshitomo et al. “Hydrogen/deuterium-exchange (H/D-Ex) of PPARg LBD in the presence of various modulators.” Protein Science: 2006. 5, 1883–1892.

9. PJK 20089 Peroxiredoxin Mutant Threonine 77 replaced with valine Threonine 77 replaced with valine

10. PJK 200810 Background Peroxiredoxin takes a catalytic pathway upon the binding of a peroxide substrate Poole.:Chapter 4: The catalytic mechanism of peroxiredoxins. In Peroxiredoxin Systems (Flohe et al.). 2007.

11. PJK 200811 Robust Peroxiredoxin Fully FoldedLocally Unfolded Wood et al. 2003

12. PJK 200812 Protocol

13. PJK 200813 Instrumentation Mass Spectrometry (MS) for biomolecular structure determination QTOF LC-MS MALDI TOF/TOF

14. PJK 200814 MALDI Results Peaks expand with longer deuterium exposure Preliminary analysis

15. PJK 200815 QTOF Results

16. PJK 200816 QTOF Results m/z = 581.83 Th Quadruple Charge State Amino acids 1-20

17. PJK 200817 QTOF Deuterium Graph

18. PJK 200818 QTOF Deuterium Graph with TCEP

19. PJK 200819 Ribbon Structure Determined From Deuterium Graph

20. PJK 200820 Future Work Replication of deuterium label graph Replication of HXMS to determine accuracy of how reduction of disulfide bridges changed structure Determine any overall conformational changes occurring with reduction of disulfide bridges

21. PJK 200821 Acknowledgements Howard Hughes Medical Institute, OSU Claudia S. Maier, PhD Kevin Ahern, PhD The Maier Laboratory Department of Biochemistry and Biophysics, OSU Department of Chemistry, OSU