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

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.

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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&params.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

PJK 2008 17

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

PJK 2008 2121

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