title: Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations.
abstract: The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324-330, 1995). In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)-substituted human insulin hexamer and the 1H NMR spectra of the Zn(II)-substituted hexamer have been carried out under a variety of ligation conditions to test the applicability of the sequential (KNF) and the half-site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d-->d transitions of the octahedral Co(II)-T-state and tetrahedral Co(II)-R-state species, and analysis of the 1H NMR spectra of T- and R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R-state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T- and R-states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer.
authors.affiliation: Department of Biochemistry, University of California, Riverside 92521, USA.
authors.forename: W E
authors.initials: WE
authors.lastname: Choi
authors.forename: D
authors.initials: D
authors.lastname: Borchardt
authors.forename: N C
authors.initials: NC
authors.lastname: Kaarsholm
authors.forename: P S
authors.initials: PS
authors.lastname: Brzovic
authors.forename: M F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D000494:Allosteric Regulation; D000838:Anions; D001704:Biopolymers; D003035:Cobalt / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000838:Anions; D001704:Biopolymers; D007328:Insulin; D003035:Cobalt