Speaker
Description
We report a comprehensive study of the structure, molecular organization, conformational heterogeneity, supramolecular packing and thermally-induced dynamics of insulin detemir, a long-lasting engineered insulin analogue obtained by removal of ThrB30 and covalent attachment of myristic acid chain to LysB29. Powder X-ray diffraction, temperature-dependent Fourier-transform infrared and Raman spectroscopy and generalized two-dimensional correlation analysis (GEN-2DCOS), together with moving-window (MW) and perturbation-correlation moving window (PCMW) 2DCOS approaches were employed to probe temperature-induced structural transformations.
The combined analyses reveal coupling between conformational changes of the protein backbone and rearrangements of the myristic acid chain, providing insights into the interplay between -helical backbone adjustments, and stabilization of hexameric assemblies. The sequence of temperature-induced structural changes was established through joint analysis of synchronous and asynchronous two-dimensional correlation spectra, enabling identification of processes associated with partial unfolding, intermolecular association and fatty acid packing. Furthermore, the applied methodology provides information on the rigidity of the disulfide bonds, their rearrangements in the course of aggregation, as well as on the propagation of conformational changes through the molecule.
The results demonstrate how conformational adaptations of the insulin backbone are linked to intermolecular self-assembly and crystallization phenomena, highlighting the utility of advanced two-dimensional correlation approaches for elucidating complex relationships between structure and dynamics in engineered protein pharmaceuticals.
Keywords: insulin detemir, protein conformational dynamics, self-association, two-dimensional correlation spectroscopy (2DCOS), moving-window (MW) 2DCOS, perturbation-correlation MW 2D COS, temperature-dependent structural dynamics.