Abstract
We have analyzed the existence of obligatory steps in the folding reaction of the α-spectrin SH3 domain by mutating Asp 48 (D48G), which is at position i+3 of an isolated two-residue type II' β-turn. Calorimetry and X-ray analysis show an entropic stabilizing effect resulting from local changes at the dihedral angles of the β-turn. Kinetic analysis of D48G shows that this β-turn is fully formed in the transition state, while there is no evidence of its formation in an isolated fragment. Introduction of several mutations in the D48G protein reveals that the local stabilization has not significantly altered the transition state ensemble. All these results, together with previous analysis of other α-spectrin and src SH3 mutants, indicate that: (i) in the folding reaction there could be obligatory steps which are not necessarily part of the folding nucleus; (ii) transition state ensembles in β-sheet proteins could be quite defined and conformationally restricted ('mechanic folding nucleus'); and (iii) transition state ensembles in some proteins could be evolutionarily conserved.
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Acknowledgements
We thank I. Angrand for help with mutagenesis and molecular biology, G. Wallon and C. Vega for invaluable help with crystallographic techniques, and F. J. Blanco and M. Ramirez-Alvarado in NMR processing. We are also grateful to P. L. Mateo for the generous offer of calorimetric techniques. We are grateful to D. Baker for providing us with a copy of his manuscript regarding the analysis of the src SH3 domain prior to publication, as well as allowing us to discuss their unpublished results. J.C.M. is supported by a TMR fellowship from the EU. M.T.P. is supported by an EU biotechnology grant.
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Martinez, J., Pisabarro, M. & Serrano, L. Obligatory steps in protein folding and the conformational diversity of the transition state. Nat Struct Mol Biol 5, 721–729 (1998). https://doi.org/10.1038/1418
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DOI: https://doi.org/10.1038/1418
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