Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues.
To elucidate the kinetic importance of structural intermediates in single-domain proteins, we measured the effect of solution conditions and amino-acid changes at a central core residue of ubiquitin (Val 26) on the kinetics of folding and unfolding. Kinetic analysis in terms of a sequential three-state mechanism provides insight into the contribution of specific interactions within the ubiquitin core to the structural stability of the native and intermediate states. The observations that disruptive mutations and/or addition of denaturants result in an apparent two-state folding process with slower rates is explained by the destabilization of a partially folded intermediate, which is in rapid equilibrium with unfolded states. The model predicts that under sufficiently stabilizing conditions kinetic intermediates may become populated even for proteins showing apparent two-state kinetics.
Khorasanizadeh
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Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Peters
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Roder
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D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism; D014633:Valine / Q000502:physiology
D014452:Ubiquitins; D014633:Valine