Spermwhale myoglobin

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Sperm-whale myoglobin
HEME
1MBO
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Mb folding intermediates
Apo-Mb
Holo-Mb
A partially folded form of holo-Myoglobin
(50 isopropanol)
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Hydrogen-exchange/MS

• The principle of hydrogen exchange experiments
• Kinetic and equilibrium experiments
• Complementarity of NMR and MS
• Site-specific information by HX/fragmentation/MS
• Examples
• Time-resolved ESI-MS

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Hydrogen exchange
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Intrinsic hydrogen-exchange rates
gtNH OD- ? gtND OH-
Secondary structure and burial Decrease the
exchange rate by several orders of magnitude
HX in combination with NMR and/or MS gives
important information about protein folding and
stability
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EX1 or EX2 regime
k1
k3 NH(close) ltgt NH(open) gt ND(open)
k2 kobs (k1k3)/(k1k2k3)
EX2 k2 gtgt k1, k3 kobs (k1k3)/k2 keqk3 DG
-RT ln (kobs /k3) Apparent DG for local
unfolding
EX1 k3 gtgt k1, k2 kobs k1
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Equilibrium HX / MS
EX1
(Miranker et al. 1993, Science 262, 896-900)
EX2
EX2
Dry protein from D2O gt H2O pH 3.8 69 C (mainly
folded). After Dt, quench at 4 C, wash 4 C gt
ESI-MS
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Pulse labeling / MS
Deuterated lysozyme, denaturing conditions pH 5.5
Start refolding (dilution in D2O)
Pulse-labeling after variable Dt (H2O pH 10)
Quench, wash, ESI-MS
(Miranker et al. 1993, Science 262, 896-900)
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Evolution of species distribution
From complementary NMR information, a folded and
b unfolded in I
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Combine HX/MS with proteolysis for site-specific
information
HX
quench
pepsin
LC/MS
Assess extent of isotope labeling in each fragment