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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1
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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2
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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3
Alpha Helices
• Hydrogen bonding in
•
alpha helices
Limits proton-solvent
exchange
•
http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg
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4
Beta Sheet
• Plenty of surface
area for solvent
interaction
•http://cnx.org/content/m11614/latest
/beta_sheet_cartoon.JPG
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5
Background
• HX-MS to analyze protein structure
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www.hxms.com/hxms.htm
6
Secondary Structure Background
•
http://www.rcsb.org/pdb/explore/remediatedSequence.do?st
ructureId=1YF0&params.chainEntityStrategyStr=all&forceP
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ageForChain=E#chainC
7
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
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in the presence of various modulators.” Protein Science: 2006. 5, 1883–1892.
8
Peroxiredoxin Mutant
• Threonine 77
replaced with
valine
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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
0621NNS5min 1333 (8.312)
TOF MS ES+
476
812.9929
100
813.5273
812.4819
811.4835
%
814.5618
Deuterium Level
0
0621NNS1min 1335 (8.337)
TOF MS ES+
366
812.4819
812.9929
100
4
813.5273
811.5067
Relative Deuterium Level (Da)
4
%
814.0504
3
814.5618
0
0621NNS0min 1336 (8.337)
3
775.00
TOF MS ES+
365
812.5168
812.0059
813.0162
100
2
813.5505
811.5183
%
814.0620
2
814.5618
0
0621NNSControl 1340 (8.357)
1
TOF MS ES+
458
811.4951
100
812.5168
1
%
813.0510
0
1
10
100
0
m/z
810
811
812
813
814
815
816
817
818
819
820
821
Time (min)
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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
0621NNS5min 1333 (8.312)
TOF MS ES+
476
812.9929
100
813.5273
812.4819
811.4835
%
814.5618
0
0621NNS1min 1335 (8.337)
TOF MS ES+
366
812.4819
812.9929
100
813.5273
811.5067
%
814.0504
814.5618
0
0621NNS0min 1336 (8.337)
TOF MS ES+
365
812.5168
812.0059
813.0162
100
813.5505
811.5183
%
814.0620
814.5618
0
0621NNSControl 1340 (8.357)
TOF MS ES+
458
811.4951
100
812.5168
%
813.0510
0
810
811
812
813
814
PJK8152008
m/z
816
817
818
819
820
821
15
QTOF Results
Deuterium Level
• m/z = 581.83
4
Relative Deuterium Level (Da)
Th
• Quadruple
Charge State
• Amino acids
1-20
4
3
3
2
581.00
2
1
1
0
1
10
100
Time (min)
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QTOF Deuterium Graph
AhpC (T77V) Deuteration Labels of Oxidized Form
Deuterium Level
3.00
0.5"
1.00"
2.50
5.00"
10.00"
2.00
15.00"
30.00"
1.50
120.0"
1.00
0.50
0.00
1--20
21-35 36-43 51-60 61-67 68-76 88-95
96110
111122
123132
133147
177182
Peptide Fragments
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QTOF Deuterium Graph with TCEP
AhpC (T77V) Deuteration Labels at Six Labeling Time Points
6.00
1"
5"
10"
15"
20"
30"
4.00
3.00
2.00
1.00
-18
2
177
-17
6
156
-14
7
133
-13
2
123
-12
2
117
96116
96103
6887
4260
4350
3643
2135
2132
1-- 2
0
0.00
1-- 5
Deuterium Level
5.00
Peptic Fragments
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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
21
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