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The monomer–dimer equilibrium for insulin is one of the essential steps in forming the receptor-binding competent monomeric form of the hormone. Despite this importance, the thermodynamic stability, in particular for modified insulins, is quite poorly understood, in part, due to experimental difficulties. This work explores one- and two-dimensional infrared spectroscopy in the range of the amide-I band for the hydrated monomeric and dimeric wild-type hormone. It is found that for the monomer the frequency fluctuation correlation function (FFCF) and the one-dimensional infrared spectra are position sensitive. The spectra of the −CO probes at the dimerization interface (residues Phe24, Phe25, and Tyr26) split and indicate an asymmetry despite the overall (formal) point symmetry of the dimer structure. Also, the decay times of the FFCF for the same −CO probe in the monomer and the dimer can differ by up to 1 order of magnitude, for example, for residue PheB24, which is solvent exposed for the monomer but at the interface for the dimer. The spectroscopic shifts correlate approximately with the average number of hydration waters and the magnitude of the FFCF at time zero. However, this correlation is only qualitative due to the heterogeneous and highly dynamical environment. Based on density functional theory calculations, the dominant contribution for solvent-exposed −CO is found to arise from the surrounding water (∼75%), whereas the protein environment contributes considerably less. The results suggest that infrared spectroscopy is a positionally sensitive probe of insulin dimerization, in particular in conjunction with isotopic labeling of the probe.
