Structural role of the proline residues of the beta-hinge region of p13suc1 as revealed by site-directed mutagenesis and fluorescence studies.
Site-directed mutagenesis, gel filtration, and fluorescence spectroscopy approaches were used to study the molecular hinge mechanism involved in the beta-strand-exchanged dimer formation of the cyclin-dependent protein kinase regulatory subunit p13(suc1) from Schizosaccharomyces pombe. Single and double mutants of residues Pro-90 and Pro-92 (P90V, P92V, and P90V/P92V) were prepared and assayed. Substitution of Pro-90 prevented dimer formation by arm exchange. However, single point mutations did not affect the two-state unfolding transition of wild-type p13(suc1) at equilibrium (i.e., wild type, DeltaG degrees (0,un) = 7.38 +/- 0.35 kcal mol(-1), vs P90V, DeltaG degrees (0,un) = 6.71 +/- 0.18 kcal mol(-1)). On the contrary, the double mutant unfolded with a complex transition, and the reaction was best described by a three-state model (N <==> I <==> U). Resolution of the state-dependent (native vs denatured) intrinsic fluorescence decay amplitudes of p13(suc1) showed that with P90V/P92V these parameters were affected at [GuHCl] significantly less than with wild-type and single mutant proteins. Moreover, with the latter products, fluorescence quenching measurements at 1 M GuHCl revealed linear Stern-Volmer plots with quenching constants typical of tryptophan residues located in a native environment (1.6 M(-1) < K(SV) < 2.3 M(-1)). Dissimilarly, with P90V/P92V a significant deviation from linearity of the Stern-Volmer plot was obtained. Nonlinear least-squares analysis of these data resolved the significant contribution of highly solvent-accessible emitting species (K(SV) = 26 M(-1)) consistent with large exposure of the tryptophan residues. These results are compatible with the existence of an intermediate unfolding state of the double mutation product. Thus, while single residue substitution studies give support to the primary role of Pro-90 in the p13(suc1) dimer formation by domain swapping, double residue substitution studies indicate the important role of the conserved repeat, Pro-x-Pro, for the proper beta-strand spatial organization and stability.
Simeoni F, Masotti L, Neyroz P