SPECTROSCOPIC EVIDENCE FOR PREEXISTING T-STATE AND R-STATE INSULIN HEXAMER CONFORMATIONS

The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperat ive heterotropic interactions (C. R. Bloom et al., J. Mel. Biol, 245, 324-330, 1995), In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)-substituted human insulin hexa mer and the H-1 NMR spectra of the Zn(II)-substituted hexamer have bee n carried out under a variety of Ligation conditions to test the appli cability of the sequential (KNF) and the half-site reactivity (SMB) mo dels for allostery, Through spectral decomposition of the characterist ic d --> d transitions of the octahedral Co(II)-T-state and tetrahedra l Co(II)-R-state species, and analysis of the H-1 NMR spectra of T- an d R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for th e phenolic pockets, The demonstration of preexisting R-state species w ith unoccupied sites is incompatible with the principles upon which th e KNF model is based, However, the SMB model requires preexisting T- a nd R-states, This feature, and the symmetry constraints of the SMB mod el make it appropriate for describing the allosteric properties of the insulin hexamer. (C) 1996 Wiley-Liss, Inc.