Probing Aromatic\nSide Chains Reveals the Site-Specific\nMelting in the SUMO1 Molten Globule

The conventional idea that a well-defined protein structure\ngoverns\nits functions is being challenged by the evolving significance of\nconformational flexibility and disorder in influencing protein activity.\nHere, we focus on the Small Ubiquitin-like MOdifier 1 (SUMO1) protein,\na post-translational modifier, which binds various target proteins\nduring the process of SUMOylation. We present evidence supporting\nthe presence of both folded and “ordered” molten globule\n(MG) states in SUMO1 under physiological conditions. We investigate\nthe MG state using a combination of near-UV and far-UV circular dichroism\n(CD) experiments. Moreover, we dissect the information from the near-UV\nCD data to gain specific insights about the MG intermediate. This\nis achieved by mutating specific aromatic amino acids, particularly\ncreating a single-tyrosine mutant S1Y51 (by introducing Y21F and Y91F\nmutations) and a tryptophan mutant S1F66W. Spectroscopic studies of\nthe mutants as a function of temperature revealed multiple insights.\nThe transition from the folded to the MG state involves a site-specific\nloss of tertiary packing near Y51 but the region surrounding F66 retained\nmost of its tertiary contacts, suggesting an ordered MG structure.\nWe further demonstrate the increased solvent exposure of Y51 in the\nMG state by using time-resolved fluorescence and steady-state quenching\nexperiments. The observed conformational flexibility and solvent accessibility,\nparticularly around Y51 that is known to be involved in binding the\ncognate ligands such as PIASX and its peptide analogues, have biological\nand functional implications in mediating protein–protein interactions\nduring the SUMOylation process.