PJK 2008 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
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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.
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Hydrogen Exchange with Solvent
• Deuterium solvent exchanges with structural protons without change in structure (A)
• Protein unfolded in exchange process (B)
• Wales & Engen 2006.
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Alpha Helices
• Hydrogen bonding in alpha helices
• Limits proton-solvent exchange
• http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg
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• Plenty of surface area for solvent interaction
•http://cnx.org/content/m11614/latest/beta_sheet_cartoon.JPG
Beta Sheet
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Background
• HX-MS to analyze protein structure
www.hxms.com/hxms.htm
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Secondary Structure Background
• http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=1YF0¶ms.chainEntityStrategyStr=all&forcePageForChain=E#chainC
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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.
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Peroxiredoxin Mutant
• Threonine 77 Threonine 77 replaced with replaced with valinevaline
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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.
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Robust Peroxiredoxin
Fully Folded Locally Unfolded
Wood et al. 2003
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Protocol
Deuterium Level
0
1
1
2
2
3
3
4
4
1 10 100
Time (min)
Re
lati
ve
De
ute
riu
m L
ev
el
(Da
)
775.00
810 811 812 813 814 815 816 817 818 819 820 821m/z0
100
%
0
100
%
0
100
%
0
100
%
0621NNS5min 1333 (8.312) TOF MS ES+ 476812.9929
812.4819
811.4835
813.5273
814.5618
0621NNS1min 1335 (8.337) TOF MS ES+ 366812.4819
811.5067
812.9929813.5273
814.0504
814.5618
0621NNS0min 1336 (8.337) TOF MS ES+ 365812.5168
812.0059
811.5183
813.0162
813.5505
814.0620
814.5618
0621NNSControl 1340 (8.357) TOF MS ES+ 458811.4951
812.5168
813.0510
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Instrumentation
• Mass Spectrometry (MS) for biomolecular structure determination
•QTOF LC-MS
•MALDI TOF/TOF
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MALDI Results
• Peaks expand with longer deuterium exposure
• Preliminary analysis
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QTOF Results
810 811 812 813 814 815 816 817 818 819 820 821m/z0
100
%
0
100
%
0
100
%
0
100
%
0621NNS5min 1333 (8.312) TOF MS ES+ 476812.9929
812.4819
811.4835
813.5273
814.5618
0621NNS1min 1335 (8.337) TOF MS ES+ 366812.4819
811.5067
812.9929813.5273
814.0504
814.5618
0621NNS0min 1336 (8.337) TOF MS ES+ 365812.5168
812.0059
811.5183
813.0162
813.5505
814.0620
814.5618
0621NNSControl 1340 (8.357) TOF MS ES+ 458811.4951
812.5168
813.0510
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QTOF Results
• m/z = 581.83 Th
• Quadruple Charge State
• Amino acids 1-20
Deuterium Level
0
1
1
2
2
3
3
4
4
1 10 100
Time (min)
Rel
ativ
e D
eute
riu
m L
evel
(D
a)581.00
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QTOF Deuterium Graph
AhpC (T77V) Deuteration Labels of Oxidized Form
0.00
0.50
1.00
1.50
2.00
2.50
3.00
1--20 21-35 36-43 51-60 61-67 68-76 88-95 96-110
111-122
123-132
133-147
177-182
Peptide Fragments
De
ute
riu
m L
ev
el
0.5"
1.00"
5.00"
10.00"
15.00"
30.00"
120.0"
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QTOF Deuterium Graph with TCEP
AhpC (T77V) Deuteration Labels at Six Labeling Time Points
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Peptic Fragments
De
ute
riu
m L
ev
el
1"
5"
10"
15"
20"
30"
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Ribbon Structure Determined From Deuterium Graph
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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
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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