Molecular Determinants of a Native-State Prolyl Isomerization

Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a beta hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol(-1) of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded beta sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the beta strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the beta sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins.