Combining High-Pressure NMR and Geometrical Sampling to Obtain a Full Topological Description of Protein Folding Landscapes: Application to the Folding of Two MAX Effectors from Magnaporthe oryzae

Despite advances in experimental and computational methods, the mechanisms by which an unstructured polypeptide chain regains its unique three-dimensional structure remains one of the main puzzling questions in biology. Single-molecule techniques, ultra-fast perturbation and detection approaches and improvement in all-atom and coarse-grained simulation methods have greatly deepened our understanding of protein folding and the effects of environmental factors on folding landscape. However, a major challenge remains the detailed characterization of the protein folding landscape. Here, we used high hydrostatic pressure 2D NMR spectroscopy to obtain high-resolution experimental structural information in a site-specific manner across the polypeptide sequence and along the folding reaction coordinate. We used this residue-specific information to constrain Cyana3 calculations, in order to obtain a topological description of the entire folding landscape. This approach was used to describe the conformers populating the folding landscape of two small globular proteins, AVR-Pia and AVR-Pib, that belong to the structurally conserved but sequence-unrelated MAX effectors superfamily. Comparing the two folding landscapes, we found that, in spite of their divergent sequences, the folding pathway of these two proteins involves a similar, inescapable, folding intermediate, even if, statistically, the routes used are different.

Automated summary

Despite advances in experimental and computational methods, the mechanisms of protein folding remain elusive. This study used high hydrostatic pressure 2D NMR spectroscopy and Cyana3 calculations to characterize the folding landscape of two unrelated proteins. It found that despite different sequences, both proteins follow a similar folding pathway involving a common intermediate.