Ligand-induced conformational change in the minimized insulin receptor

Within the class of insulin and insulin-like growth factor receptors, detai led information about the molecular recognition event at the hormone-recept or interface is limited by the absence of suitable co-crystals. We describe the use of a biologically active insulin derivative labeled with the NBD f luorophore (B29NBD-insulin) to characterize the mechanism of reversible 1:1 complex formation with a fragment of the insulin receptor ectodomain. The accompanying 40 % increase in the fluorescence quantum yield of the label p rovides the basis for a dynamic study of the hormone-receptor binding event . Stopped-flow fluorescence experiments show that the kinetics of complex f ormation are biphasic comprising a bimolecular binding event followed by a conformational change. Displacement with excess unlabeled insulin gave mono phasic kinetics of dissociation. The rate data are rationalized in terms of available experiments on mutant receptors and the X-ray structure of a non -binding fragment of the receptor of the homologous insulin-like growth fac tor (IGF-1). (C) 2000 Academic Press.