Stability and folding of the cell cycle regulatory protein, p13suc1

p13(suc1) (suc1) is a member of the CDC28 kinase specific family of cell cycle regulatory proteins that bind to the cyclin-dependent kinase CDK2 and regulate its activity. suc1 has two distinct conformational and assembly states, a compact globular monomer and a beta strand-exchanged dimer. The dimerisation is an example of domain-swapping, and is mediated by a molecular hinge mechanism that is conserved across the entire CKS family. It has been proposed that the function of suc1 may be modulated by the dimerisation process with monomer-dimer switching occurring in response to a change in the cell environment. We have investigated the stability and folding of suc1 as a first step in determining the mechanism and functional role of the strand exchange. Suc1 unfolds reversibly at equilibrium in a two-state manner with a free energy of unfolding of 7.2 kcal mol(-1). The kinetics of folding and unfolding are complex, and double-jump stopped-flow methods revealed that there are at least three parallel folding pathways arising from distinct unfolded and partly folded, intermediate states. The major population of unfolded species fold rapidly according to a three-state mechanism, D-1 - > I-1 - > N, with a rate constant for the formation of native species, N, from the intermediate, I-1, of 65 s(-1) in water. Two minor populations of unfolded molecules fold more slowly. Folding of one population is limited by proline isomerisation in a partly folded state, and some expansion of the protein is required for isomerisation to occur. The other population could be assigned to rate-limiting isomerisation of the peptidyl-proline bond of residue 90, which is located in the molecular hinge. A minor, fast phase was detected in the unfolding kinetics that corresponds to unfolding of a small population of a distinct native-like form. Heterogeneity was removed upon mutation of Pro90 to Ala. The unfolding kinetics of the strand-exchanged dimer were also investigated and showed that the dimer unfolds at the same rate as the monomer. (C) 1998 Academic Press.