Enhancing protein stability with extended disulfide bonds

Significance This work describes a facile system for incorporating noncanonical amino acids containing long side-chain thiols using an expanded genetic code. These amino acids begin to overcome the distance and geometric constraints of the cysteine disulfide and can pair with cysteines to cross-link more remote sites in proteins. To demonstrate this notion, we constructed a library of random β-lactamase mutants containing these noncanonical amino acids and grew them at nonpermissive temperatures. We identified a mutant enzyme that is cross-linked by one such extended disulfide bond that has significantly enhanced thermal stability. This study suggests that an expanded set of amino acid building blocks can provide novel solutions to evolutionary challenges.