Elucidating physical mechanisms with statistical confidence from molecular dynamics simulations can be challenging owing to the many degrees of freedom that contribute to collective motions. To address this issue, we recently introduced a dynamical Galerkin approximation (DGA) [ThiedeE. H. Thiede, E. H. J. Chem. Phys.2019150244111], in which chemical kinetic statistics that satisfy equations of dynamical operators are represented by a basis expansion. Here, we reformulate this approach, clarifying (and reducing) the dependence on the choice of lag time. We present a new projection of the reactive current onto collective variables and provide improved estimators for rates and committors. We also present simple procedures for constructing suitable smoothly varying basis functions from arbitrary molecular features. To evaluate estimators and basis sets numerically, we generate and carefully validate a data set of short trajectories for the unfolding and folding of the trp-cage miniprotein, a well-studied system. Our analysis demonstrates a comprehensive strategy for characterizing reaction pathways quantitatively.

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 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.

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.

Therapeutic preparations of insulin often contain phenolic molecules, which can impact both pharmacokinetics and shelf life. Thus, understanding the interactions of insulin and phenolic molecules can aid in designing improved therapeutics. In this study, we use molecular dynamics to investigate phenol release from the insulin hexamer. Leveraging recent advances in methods for analyzing molecular dynamics data, we expand on existing simulation studies to identify and quantitatively characterize six phenol binding/unbinding pathways for wild-type and A10 Ile → Val and B13 Glu → Gln mutant insulins. A number of these pathways involve large-scale opening of the primary escape channel, suggesting that the hexamer is much more dynamic than previously appreciated. We show that phenol unbinding is a multipathway process, with no single pathway representing more than 50% of the reactive current and all pathways representing at least 10%. We use the mutant simulations to show how the contributions of specific pathways can be rationally manipulated. Predicting the net effects of mutations is more challenging because the kinetics depend on all of the pathways, demanding quantitatively accurate simulations and experiments.

Experimental evidence suggests that monomeric insulin exhibits significant conformational heterogeneity, and modifications of apparently disordered regions affect both biological activity and the longevity of pharmaceutical formulations, presumably through receptor binding and fibrillation/degradation, respectively. However, a microscopic understanding of conformational heterogeneity has been lacking. Here, we integrate all-atom molecular dynamics simulations with an analysis pipeline to investigate the structural ensemble of human insulin monomers. We find that 60% of the structures present at least one of the following elements of disorder: melting of the A-chain N-terminal helix, detachment of the B-chain N-terminus, and detachment of the B-chain C-terminus. We also observe partial melting and extension of the B-chain helix and significant conformational heterogeneity in the region containing the B-chain β-turn. We then estimate hydrogen-exchange protection factors for the sampled ensemble and find them in line with experimental results for KP-insulin, although the simulations underestimate the importance of unfolded states. Our results help explain the ready exchange of specific amide sites that appear to be protected in crystal structures. Finally, we discuss the implications for insulin function and stability.