Experimental comparison of energy landscape features of ubiquitin family proteins

Abstract

Abstract Small ubiquitin‐related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence‐structure‐stability‐function relationship. Here, we used chemical denaturation in comparing the folding‐unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W‐pseudo wild‐type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant‐dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two‐state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow‐down their folding rates. The denaturant‐dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent‐accessible surface area, as quantified by the Tanford β u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding‐unfolding rates within a structurally homologous protein family.