A single buried cysteine acts as a hydrophobic stabilizer of a folding intermediate and transition state in the MATH domain of SPOP

Abstract Cysteine is a highly conserved amino acid with diverse roles in protein function. Whilst its role in the formation of disulfide bridges is well characterized, the contribution of isolated cysteines in protein folding is by and large unexplored. Here we investigate the impact of cysteine residues on the folding pathway of the MATH domain in the SPOP protein by comparing wild‐type and serine mutants. Through kinetic analyses, we demonstrate that a buried cysteine residue stabilizes both an early folding intermediate and the main transition state. Most notably, such effects are disrupted upon substitution with serine but preserved with alanine. These findings suggest that, in certain structural contexts, cysteine behaves as a hydrophobic rather than a polar residue. Our results challenge the traditional classification of cysteine as a polar amino acid and highlight its unique contributions to protein folding, with implications for protein engineering and structural biology.