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

The interaction between monomeric insulin and monosaccharides has been inve stigated through circular dichroism, fluorescence spectroscopy and two dime nsional nuclear magnetic resonance. CD spectra indicate that D-glucose inte racts with monomeric insulin whereas D-galactose. D-mannose and 2-deoxy-D-g lucose have a lower effect. Fluorescence emission was quenched at sugar con centrations of 5-10 mM. Titration with the different sugars produces a quen ching of the tyrosine spectrum from which a binding free energy value for t he 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 beta form and the monomeric insulin. Further, NMR total correlation spectra expe riments revealed that the hormone is in the monomeric form and that upon ad dition of glucose no aggregation occurs. The interaction does not involve r elevant changes in the secondary structure of insulin suggesting that the i nteraction occur at the side chain level. Molecular dynamics simulations an d modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additio nal informations to locate the putative binding sites Of D-glucose to insul in. (C) 2001 Elsevier Science B.V.. All rights reserved.