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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 strand. This salt bridge is highly important for stabilization of the ubiquitin fold. However, small ubiquitin related modifier 2 (SUMO2) is an exception in the family as it lacks the above-mentioned salt bridge. SUMO2 shares only ~16% sequence similarity with ubiquitin, but the native structures are similar for both the proteins. Hydrophobic cores of SUMO2 and ubiquitin are maintained by conserved hydrophobic residues while charge topology of SUMO2 and ubiquitin differs significantly. We therefore wanted to investigate how similar or different the folding/unfolding pathway(s) of both proteins will be owing to their stark sequence dissimilarity.
Firstly, temperature was used to perturb the native (N) state of SUMO2 and access states that were more flexible than the N state. Careful investigation revealed that such temperature induced flexible states are very similar to the N state in terms of secondary structural content but have undergone a considerable decrease in tertiary structure as revealed by near UV circular dichroism (CD) and time-resolved fluorescence anisotropy. Ubiquitin on the other hand did not show increased flexibility when treated to a similar temperature perturbation. Detailed analysis carried out to ascertain the interactions hindering temperature induced flexibility of ubiquitin revealed that the above-mentioned salt bridge which is conserved in ubiquitin family proteins but absent in SUMO2 made all the difference. On disrupting the salt bridge, we were able to create mutants of ubiquitin that were as flexible as SUMO2. Our studies on Ubiquitin and SUMO2 suggest that the conserved salt bridge of Ubiquitin family proteins plays an important role in determining conformational flexibility
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