Microsecond Protein Folding Through a Compact Transition State

Author： Burton R.E. Huang G.S. Daugherty M.A. Fullbright P.W. Oas T.G.

ISSN： 0022-2836

Source： Journal of Molecular Biology, Vol.263, Iss.2, 1996-10, pp. : 311-322

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Abstract

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda 6 - 85 , a truncated, monomeric form of the N-terminal domain of lambdarepressor, refolds with a lifetime of approximately 250 mus. These methods have also been applied to a thermostable lambda 6 - 85 variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln ( k f,u ) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to 0M urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 mus. The slopes of the ln ( k f,u ) versus [urea] curves ( m u and m f ) indicate that the modest Gly -< Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda 6 - 85 has a fractional accessibility ( m u /( m u - m f )) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism.