Phi-Value and NMR Structural Analysis of a Coupled Native-State Prolyl Isomerization and Conformational Protein Folding Process

Prolyl <i>cis</i>/<i>trans</i> isomerization is a rate-limiting step in protein folding, often coupling directly to the acquisition of native structure. Here, we investigated the interplay between folding and prolyl isomerization in the N2 domain of the gene-3-protein from filamentous phage fd, which adopts a native-state <i>cis</i>/<i>trans</i> equilibrium at Pro161. Using mutational and Φ-value analysis, we identified a discrete folding nucleus encompassing the β-strands surrounding Pro161. These native-like interactions form early in the folding pathway and provide the energy to shift the <i>cis/trans</i> equilibrium toward the <i>cis</i> form. Variations distant from the Pro161-loop have minimal impact on the <i>cis</i>/<i>trans</i> ratio, underscoring the spatial specificity and localized control of the isomerization process. Using NMR spectroscopy, we determined the structures for both native N2 forms. The <i>cis</i>- and <i>trans</i>-Pro161 conformations are overall identical and exhibit only slight differences around the Pro161-loop. The <i>cis</i>-conformation adopts a more compact structure with improved backbone hydrogen bonding, explaining the approximately 10 kJ·mol^-1 stability increase of the <i>cis</i> state. Our findings highlight that prolyl isomerization in the N2 domain is governed by a localized folding nucleus rather than global stability changes. This localized energetic coupling ensures that proline isomerization is not simply a passive, slow step but an integral component of the folding landscape, optimizing both the formation of native structure and the establishment of the <i>cis</i>-conformation.