Salt Bridges in Ubiquitin Determine the Protein Conformational Flexibility

Abstract

Ubiquitin family proteins are involved in a wide variety of functions, ranging from proteasomal degradation to signal transduction. Apart from their unique β-grasp fold, with 5 β-strands wrapping around an α-helix, a unique feature of them is a conserved salt bridge connecting the α-helix to a loop between the β3 and β4 strands. Here, we investigated the role of this conserved salt bridge in determining the protein conformational flexibility using ubiquitin as a model system and utilizing time-resolved fluorescence anisotropy and near-UV circular dichroism (CD). Therefore, we inserted a Trp (L43W) at the core near to the conserved salt bridge (K27-D52), as a reporter of protein's microenvironment. CD and fluorescence anisotropy showed that ubiquitin's core is conformationally rigid in the temperature range 5-55 0C. Disrupting the salt bridge by a double mutation (K27A/D52L) increased the conformational flexibility of the protein core at 5 0C, which further increased with temperature up to 55 0C. Interestingly, SUMO2 lacking such a conserved salt bridge, has recently been shown to have similar flexibility in this temperature range.1 These results suggest that the core flexibility of the salt bridge (K27A/D52L) disrupted ubiquitin resembles that of SUMO2. Furthermore, a more severe salt bridge mutation (K27M/D52L) of ubiquitin, replacing Lys with Met to mimic SUMO2, showed that the protein's core is highly flexible even at 5 0C and devoid of any tertiary structure. Our studies on ubiquitin and SUMO2 suggest that the conserved salt bridge of ubiquitin family proteins plays an important role in determining protein conformational flexibility. 1. Shrabasti Bhattacharya and Sri Rama Koti Ainavarapu. Mechanical Softening of a Small Ubiquitin-Related Modifier Protein Due to Temperature Induced Flexibility at the Core. J. Phys. Chem. B (2018), 122, p9128-9136.