Protein unfolding rates correlate as strongly as folding rates with native structure

Abstract

Although the folding rates of proteins have been studied extensively, both experimentally and theoretically, and many native state topological parameters have been proposed to correlate with or predict these rates, unfolding rates have received much less attention. Moreover, unfolding rates have generally been thought either to not relate to native topology in the same manner as folding rates, perhaps depending on different topological parameters, or to be more difficult to predict. Using a dataset of 108 proteins including two-state and multistate folders, we find that both unfolding and folding rates correlate strongly, and comparably well, with well-established measures of native topology, the absolute contact order and the long range order, with correlation coefficient values of 0.75 or higher. In addition, compared to folding rates, the absolute values of unfolding rates vary more strongly with native topology, have a larger range of values, and correlate better with thermodynamic stability. Similar trends are observed for subsets of different protein structural classes. Taken together, these results suggest that choosing a scaffold for protein engineering may require a compromise between a simple topology that will fold sufficiently quickly but also unfold quickly, and a complex topology that will unfold slowly and hence have kinetic stability, but fold slowly. These observations, together with the established role of kinetic stability in determining resistance to thermal and chemical denaturation as well as proteases, have important implications for understanding fundamental aspects of protein unfolding and folding and for protein engineering and design.

Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange

“…Instead, they found that the refolding rate constant was highly dependent on relative contact order, a measure of local and non-local contacts in a protein's native structure. Broom et al (32) corroborated their result and showed that the unfolding rate constant also correlated with contact order, although less well. They concluded, "Unfolding rates correlate better with thermodynamic stability."…”

Designed protein reveals structural determinants of extreme kinetic stability

“…ACO/LRO are measures of topological complexity based on the sequence separation of contacting residues in the folded protein. We have shown recently that the rates of protein folding and unfolding both decrease with increasing ACO/ LRO (41). LRO provides a more linear and stronger correlation and is normalized for increasing protein size, which also slows (un)folding (41,42).…”

“…We have shown recently that the rates of protein folding and unfolding both decrease with increasing ACO/ LRO (41). LRO provides a more linear and stronger correlation and is normalized for increasing protein size, which also slows (un)folding (41,42). As ACO/LRO provide just a simple measure of protein structural complexity, we used Gō models to further define the molecular origins of ThreeFoil's high barrier.…”

“…ThreeFoil folding/unfolding kinetics are extremely slow compared with other proteins. Rate constants (gray diamonds) at the transition midpoint (ln(k f ) = ln(k u )) for a large dataset of proteins (SI Appendix, Table S2) (41), are correlated with ACO (A) and LRO (B). β-trefoil proteins: ThreeFoil (orange), Symfoil (green), and Hisactophilin (blue) are highlighted.…”

“…3 A and B) (41) and between slow unfolding and SDS/protease resistance (44,46), we asked whether these resistances could be predicted from topological complexity. We conducted experiments and surveyed the literature to identify proteins with experimentally demonstrated resistance, or lack thereof, to SDS or protease.…”