Equilibrium Hydrogen Exchange Reveals Extensive Hydrogen Bonded Secondary Structure in the On-pathway Intermediate of Im7

Stanislaw A. Gorski, Cécile S. Le Duff, Andrew P Capaldi, Arnout P. Kalverda, Godfrey S. Beddard, Geoffrey R. Moore, Sheena E. Radford

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 (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.

Original languageEnglish (US)
Pages (from-to)183-193
Number of pages11
JournalJournal of Molecular Biology
Volume337
Issue number1
DOIs
StatePublished - Mar 12 2004
Externally publishedYes

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Hydrogen
Amides
Proteins
Mutant Proteins
Immunity
Amino Acids

Keywords

  • Hydrogen exchange
  • Immunity protein
  • Intermediates

ASJC Scopus subject areas

  • Virology

Cite this

Equilibrium Hydrogen Exchange Reveals Extensive Hydrogen Bonded Secondary Structure in the On-pathway Intermediate of Im7. / Gorski, Stanislaw A.; Le Duff, Cécile S.; Capaldi, Andrew P; Kalverda, Arnout P.; Beddard, Godfrey S.; Moore, Geoffrey R.; Radford, Sheena E.

In: Journal of Molecular Biology, Vol. 337, No. 1, 12.03.2004, p. 183-193.

Research output: Contribution to journalArticle

Gorski, Stanislaw A. ; Le Duff, Cécile S. ; Capaldi, Andrew P ; Kalverda, Arnout P. ; Beddard, Godfrey S. ; Moore, Geoffrey R. ; Radford, Sheena E. / Equilibrium Hydrogen Exchange Reveals Extensive Hydrogen Bonded Secondary Structure in the On-pathway Intermediate of Im7. In: Journal of Molecular Biology. 2004 ; Vol. 337, No. 1. pp. 183-193.
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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 (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.",
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AU - Capaldi, Andrew P

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AU - Beddard, Godfrey S.

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