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Using HX-MS to Determine Protein Structure of AhpC 2, a Peroxiredoxin

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. Hypothesis.

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Using HX-MS to Determine Protein Structure of AhpC 2, a Peroxiredoxin

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  1. 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 PJK 2008

  2. 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. PJK 2008

  3. Hydrogen Exchange with Solvent Deuterium solvent exchanges with structural protons without change in structure (A) Protein unfolded in exchange process (B) Wales & Engen 2006. PJK 2008

  4. Alpha Helices Hydrogen bonding in alpha helices Limits proton-solvent exchange http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg PJK 2008

  5. Beta Sheet • Plenty of surface area for solvent interaction • http://cnx.org/content/m11614/latest/beta_sheet_cartoon.JPG PJK 2008

  6. Background HX-MS to analyze protein structure www.hxms.com/hxms.htm PJK 2008

  7. Secondary Structure Background http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=1YF0&params.chainEntityStrategyStr=all&forcePageForChain=E#chainC PJK 2008

  8. 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. PJK 2008

  9. Peroxiredoxin Mutant Threonine 77 replaced with valine PJK 2008

  10. 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. PJK 2008

  11. Robust Peroxiredoxin Fully Folded Locally Unfolded Wood et al. 2003 PJK 2008

  12. Protocol PJK 2008

  13. Instrumentation Mass Spectrometry (MS) for biomolecular structure determination • QTOF LC-MS • MALDI TOF/TOF PJK 2008

  14. MALDI Results Peaks expand with longer deuterium exposure Preliminary analysis PJK 2008

  15. QTOF Results PJK 2008

  16. QTOF Results m/z = 581.83 Th Quadruple Charge State Amino acids 1-20 PJK 2008

  17. QTOF Deuterium Graph PJK 2008

  18. QTOF Deuterium Graph with TCEP PJK 2008

  19. Ribbon Structure Determined From Deuterium Graph PJK 2008

  20. 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 PJK 2008

  21. 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 21 PJK 2008

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