Experimental comparison of energy landscape features of ubiquitin family proteins.

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.

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