Insulin dissociates by diverse mechanisms of coupled unfolding and unbinding

The protein hormone insulin exists in various oligomeric forms, and a key step in binding its cellular receptor is dissociation of the dimer. This dissociation process and its corresponding association process have come to serve as a paradigms of coupled (un)folding and (un)binding more generally. Despite its fundamental and practical importance, the mechanism of insulin dimer dissociation remains poorly understood. Here, we use molecular dynamics simulations, leveraging recent developments in umbrella sampling, to characterize the energetic and structural features of dissociation in unprecedented detail. We find that the dissociation is inherently multipathway with limiting behaviors corresponding to conformational selection and induced fit, the two prototypical mechanisms of coupled folding and binding. Along one limiting path, the dissociation leads to detachment of the C-terminal segment of the insulin B chain from the protein core, a feature believed to be essential for receptor binding. We simulate IR spectroscopy experiments to aid in interpreting current experiments and identify sites where isotopic labeling can be most effective for distinguishing the contributions of the limiting mechanisms.