Computational IR Spectroscopy of Insulin Dimer Structure and Conformational Heterogeneity.

We have investigated the structure and conformational dynamics of insulin dimer using a Markov state model (MSM) built from extensive unbiased atomistic molecular dynamics simulations and performed infrared spectral simulations of the insulin MSM to describe how structural variation within the dimer can be experimentally resolved. Our model reveals two significant conformations to the dimer: a dominant native state consistent with other experimental structures of the dimer and a twisted state with a structure that appears to reflect a ∼55° clockwise rotation of the native dimer interface. The twisted state primarily influences the contacts involving the C-terminus of insulin's B chain, shifting the registry of its intermolecular hydrogen bonds and reorganizing its side-chain packing. The MSM kinetics predict that these configurations exchange on a 14 μs time scale, largely passing through two Markov states with a solvated dimer interface. Computational amide I spectroscopy of site-specifically 13C18O labeled amides indicates that the native and twisted conformation can be distinguished through a series of single and dual labels involving the B24F, B25F, and B26Y residues. Additional structural heterogeneity and disorder is observed within the native and twisted states, and amide I spectroscopy can also be used to gain insight into this variation. This study will provide important interpretive tools for IR spectroscopic investigations of insulin structure and transient IR kinetics experiments studying the conformational dynamics of insulin dimer.