Disulfide Bonds are Allosteric Regulator of Mechanical Stability

Disulfide bonds are known to stabilize proteins against perturbations such as temperature or denaturants. Since mechanical force is the most common protein denaturant in vivo, there is increasing interest in the role that disulfide bonds have in the mechanical unfolding of proteins. For instance, disulfide bonds reduce the contour length of stretched proteins by limiting the extensibility up to the covalently linked cysteines. However very little is known about the effect of native disulfide bonds in the strength of structural clamps that determine mechanical stability.Here, we use single molecule atomic force spectroscopy to study the mechanical effects of disulfide bonding in immunoglobulin domains (Ig) that are constitutively under force.We observe that the formation of native disulfide bonds in immunoglobulin domains triggers drastic differences in the rupture forces, although the two cognate cysteines do not link together the β-strands of the mechanical clamp motif. In the case of bacterial gram-negative pilin FimH, disulfide increases the most probable unfolding force from 297 pN to 425 pN at 400nm/s. In contrast, the disulfide in the 69th immunoglobulin domain of human titin decreases the unfolding force at a pulling rate of 1200nm/s from 271 pN to 195 pN. We observe that both in titin and in the Fim pilus, the disulfide bonds are remarkably conserved along the entire stretched molecular architecture. Hence, many Igs in elastic segment of titin and in all Fim Igs are predicted to change mechanical stability depending of their oxidation state. Our results suggest that native disulfide bonds alter allosterically the transition state and modulate the internal flexibility of the stressed protein prior the breakage of its mechanical clamp motif.