Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7

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Item Type:	Article
Title:	Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7
Creators Name:	Gorski, S.A., Le Duff, C.S., Capaldi, A.P., Kalverda, A.P., Beddard, G.S., Moore, G.R. and Radford, S.E.
Abstract:	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 (ΔΔG(ui)=4.4kJ/mol) but the native state is only slightly destabilised (ΔΔG(ui)=1.8kJ/mol) at 10 °C in (2(H(2)O, pH(∗) 7.0 containing 0.4 M Na(2)SO(4), 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.
Keywords:	Hydrogen Exchange, Intermediates, Immunity Protein
Source:	Journal of Molecular Biology
ISSN:	0022-2836
Publisher:	Elsevier
Volume:	337
Number:	1
Page Range:	183-193
Date:	12 March 2004
Official Publication:	https://doi.org/10.1016/j.jmb.2004.01.004
PubMed:	View item in PubMed

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