TY - JOUR
T1 - Equilibrium Hydrogen Exchange Reveals Extensive Hydrogen Bonded Secondary Structure in the On-pathway Intermediate of Im7
AU - Gorski, Stanislaw A.
AU - Le Duff, Cécile S.
AU - Capaldi, Andrew P.
AU - Kalverda, Arnout P.
AU - Beddard, Godfrey S.
AU - Moore, Geoffrey R.
AU - Radford, Sheena E.
N1 - Funding Information:
We thank Alison Ashcroft for performing mass spectrometry experiments, Gary Thompson, Alexander Geisen and members of the Homans labs for help with NMR and Colin Kleanthous (York) for provision of doubly labelled Im7. We also thank Colin Kleanthous and Susan Marqusee for helpful discussion and members of the Radford laboratory for their input throughout this work. We acknowledge with thanks the Wellcome Trust, the BBSRC and the University of Leeds for financial support. S.A.G., A.P.C., A.K. and S.E.R. are members of the Astbury Centre for Structural Molecular Biology, which is part of the North of England Structural Biology Centre and is funded by the BBSRC. S.E.R. is a BBSRC Professorial fellow. We also thank the Wellcome Trust for support of the UEA Colicin Research Group and the BBSRC for a studentship (to C.S.L.D.).
PY - 2004/3/12
Y1 - 2004/3/12
N2 - The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (ΔΔGui=4.4kJ/mol), but the native state is only slightly destabilised (ΔΔG nu=1.8kJ/mol) at 10°C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Φ-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.
AB - The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (ΔΔGui=4.4kJ/mol), but the native state is only slightly destabilised (ΔΔG nu=1.8kJ/mol) at 10°C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Φ-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.
KW - Hydrogen exchange
KW - Immunity protein
KW - Intermediates
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U2 - 10.1016/j.jmb.2004.01.004
DO - 10.1016/j.jmb.2004.01.004
M3 - Article
C2 - 15001361
AN - SCOPUS:1442351134
SN - 0022-2836
VL - 337
SP - 183
EP - 193
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -