Connecting Protein\nMillisecond Conformational Dynamics\nto Protein Thermal Stability

The stability of\nprotein folded states is crucial for\nits function,\nyet the relationship with the protein sequence remains poorly understood.\nPrior studies have focused on the amino acid composition and thermodynamic\ncouplings within a single folded conformation, overlooking the potential\ncontribution of protein dynamics. Here, we address this gap by systematically\nanalyzing the impact of alanine mutations in the C-terminal β-strand\n(β5) of ubiquitin, a model protein exhibiting millisecond timescale\ninterconversion between two conformational states differing in the\nβ5 position. Our findings unveil a negative correlation between\nmillisecond dynamics and thermal stability, with alanine substitutions\nat seemingly flexible C-terminal residues significantly enhancing\nthermostability. Integrating spectroscopic and computational approaches,\nwe demonstrate that the thermally unfolded state retains a substantial\nsecondary structure but lacks β5 engagement, recapitulating\nthe transition state for millisecond dynamics. Thus, alanine mutations\nthat modulate the stabilities of the folded states with respect to\nthe partially unfolded state impact both the dynamics and stability.\nOur findings underscore the importance of conformational dynamics\nwith implications for protein engineering and design.