Hydrogen exchange

1
Hydrogen exchange proteins
2
from Wüthrich, 1986, p. 24
Eigen kD 10?pKa ktr
10?pKa 1
diffusion control rate kD 1010 M-1 s-1 at 25 C
3
Hydrogen exchange in proteins (Hvidt Nielsen,
1966)
Hi
Hi
native
open
For each hydrogen i N(H)
O(H) O(D) N(D)
kex D2O
4
For conditions favoring the folded state, kcl gtgt
kop, and kobs kop kex catalyst / (kcl kex
catalyst) This equation simplifies in two
limiting cases EX2 limit where rate of
catalysis is low kcl gtgt kex catalyst kobs
(kop / kcl) kex Kop kexcatalyst EX1
limit where rate is independent of
catalyst kobs kop
yields equilibrium constant for unfolding if kex
known
yields rate of protein opening
5
EX1 (apparent unimolecular) k1 opening rate
kobs
EX2 (bimolecular)
kex catalyst
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In proteins under most conditions the EX2 (slow
exchange limit) holds, and one can estimate the
standard free energy change of the opening
reaction from ?Goop - RT ln (kobs /
kex) Classes of exchangeable hydrogens Fastest
exchange rate Fully exposed sites Intermediate
rate Sites exposed by local unfolding Slowest
rate Rates correlate with thermal stability
global unfolding process
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Exchange studies allow one to pinpoint local
changes in free energy Case study hydrogen
exchange of staphylococcal nuclease in its
unligated (SNase) and ternary complex
(SNaseCa2pdTp) states Loh et al. (1993)
Biochemistry 32, 11022
8
Expected peptide NH exchange rates from the
denatured state (kex) are available from the
literature. Roder et al. (1985) Biochemistry 24,
7407 kex kDD kODOD- At 0 ?C, kD
3.47 min-1 M-1 kOD 2.77 ? 1010 min-1
M-1 Corrections are made for nearest neighbors
effects Molday et al., (1972) Biochemistry 11,
150 Slower exchange at Val Robertson Baldwin
(1991) Biochemistry 30, 9907
9
Temperature corrections are made with ?HD 15
kcal mol-1 ?HOD 2.5 kcal mol-1 Englander
Poulsen (1969) Biopolymers 7, 379
10
staphylococcal nuclease ternary complex
11
staphylococcal nuclease freshly dissolved in
D2O 1H- 15N HSQC
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(No Transcript)
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unligated
ligated
ratios for unligated and ligated expressed as
energy differences
14
unligated
ligated
ratios for unligated and ligated expressed as
energy differences
15
white exchange within dead time of expt
gray intermediate exchange
black slowest exchange
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black spheres residues with abnormally
large ??Guo values
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- RT ln Kapp ?Gu m urea
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• Summary of results with SNase
• almost all amides that are exposed to solvent
  exchange within the dead time
• an exception is Val39 which is exposed to solvent
  but which has a large protection factor
• other such anomalously protected NHs
• Lys55 of OMTKY3 (Robertson et al., 1988)
• Cys64 of hen egg-white lysozyme (Radford et al.,
  1992)
• amides showing the largest protection factors are
  in ?-sheet and a ?-helix
• as expected, N-terminal residues of ?-helices
  show lower protection than rest of helix (because
  they lack H-bond acceptors)

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Comparison of unfolding free energies from
hydrogen exchange and urea denaturation
experiments
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M. Kainosho use of isotope effect to get at fast
backbone hydrogen exchange
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(No Transcript)
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Hydrogen exchange nucleic acids
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In nucleic acids, exchange is always in the
faster exchange (catalyzed) limit known
quantity of ammonia is frequently added as
catalyst conditions for lifetime (?ex) of 1 s
10 ?M NH3, 1 mM phosphate, pH 7.5 M. Gueron
24
Lifetimes of imino hydrogens in nucleic acids pH
dependence
25
Lifetimes of imino hydrogens in nucleic
acids dependence on the pKa of the buffer
log ?ex
7 12
buffer pKa
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In mononucleotides, the important factor is the
difference between the pKa of the hydrogen at the
site of exchange (T) and the pKa of the buffer
(NH3) T-NH ND3 T-ND ND2H goes
through T-N- (ND3H) intermediate ?ex-1 ktr
kr NH3 ? 1 10 exp (pKT - pKNH3)-1
27
from Wüthrich, 1986, p. 24
28
With nucleic acid bases in duplexes, hydrogen
exchange occurs only through the rupture of the
H-bond and exposure of the hydrogen to
solvent T-NH . NA
T-NH NA
ktr
?ex ?o (?mono / catalyst) ? (1/Kdis)
open-limited lifetime