Intermediates control domain swapping during folding of p13suc1.
The 13-kDa protein p13(suc1) has two folded states, a monomer and a structurally similar domain-swapped dimer formed by exchange of a beta-strand. The refolding reaction of p13(suc1) is multiphasic, and in this paper we analyze the kinetics as a function of denaturant and protein concentration and compare the behavior of wild type and a set of mutants previously designed with dimerization propensities that span 9 orders of magnitude. We show that the folding reactions of wild type and all mutants produce the monomer predominantly despite their very different equilibrium behavior. However, the addition of low concentrations of denaturant in the refolding buffer leads to thermodynamic control of the folding reaction with products that correspond to the wild type and mutant equilibrium dimerization propensities. We present evidence that the kinetic control in the absence of urea arises because of the population of the folding intermediates. Intermediates are usually considered to be detrimental to folding because they slow down the reaction; however, our work shows that intermediates buffer the monomeric folding pathway against the effect of mutations that favor the nonfunctional, dimeric state at equilibrium.
Rousseau
Frederic
F
Medical Research Council Centre for Protein Engineering, Cambridge University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Schymkowitz
Joost W H
JW
Wilkinson
Hannah R
HR
Itzhaki
Laura S
LS
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D007700:Kinetics; D016296:Mutagenesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe