A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease

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Abstract

Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dΔG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand β-barrel of wild type and E75A and D77A, two mutants with reduced values of mGuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the β-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.

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Figure 1: RasMol diagram of the crystal structure of staphylococcal nuclease (1snc)10.
Figure 2: The 1H-15N correlation spectra of two forms of staphylococcal nuclease.
Figure 3: Plots of the amide exchange protection factors as a function of residue position.
Figure 4: Superimposed kinetic traces of refolding.
Figure 5: Guanidine hydrochloride denaturation of the E75A mutant monitored by NMR.
Figure 6: A schematic diagram showing the correspondence between the m value (mGuHCl) and residual structure in the denatured state.

Notes

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    This online version has been updated to correct a problem with references throughout the article. An updated PDF version will appear in a future version.

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Acknowledgements

We thank T. Sosnick and W. Englander for providing a spreadsheet for calculating intrinsic exchange rate, H. Roder, D. Barrick, and S. Mori for helpful discussions. This work was supported by The National Institutes of Health.

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Correspondence to David Shortle.

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