Spectroscopic and molecular dynamics simulation studies of the interaction of insulin with glucose

Abstract

The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that d-glucose interacts with monomeric insulin whereas d-galactose, d-mannose and 2-deoxy- d-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5–10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin–sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of d-glucose in the β form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of d-glucose to insulin.