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,

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Presentation transcript:

PJK 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 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 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 Alpha Helices Hydrogen bonding in alpha helices Limits proton-solvent exchange

PJK Plenty of surface area for solvent interaction /beta_sheet_cartoon.JPG Beta Sheet

PJK Background HX-MS to analyze protein structure

PJK Secondary Structure Background ructureId=1YF0&params.chainEntityStrategyStr=all&forceP ageForChain=E#chainC

PJK 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: , 1883–1892.

PJK Peroxiredoxin Mutant Threonine 77 replaced with valine Threonine 77 replaced with valine

PJK 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.)

PJK Robust Peroxiredoxin Fully FoldedLocally Unfolded Wood et al. 2003

PJK Protocol

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

PJK MALDI Results Peaks expand with longer deuterium exposure Preliminary analysis

PJK QTOF Results

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

PJK QTOF Deuterium Graph

PJK QTOF Deuterium Graph with TCEP

PJK Ribbon Structure Determined From Deuterium Graph

PJK 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 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