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.

Structural signatures of the complex formed between 3‐nitro‐4‐hydroxybenzoate and the Zn(II)‐substituted R6 insulin hexamer

“…The dynamics of 3N4H binding were examined by analysis of the line widths of the 1 H NMR signals for the free and bound species. When 3N4H is present in solutions of the Zn 2+ -R 6 insulin hexamer, two sets of ligand resonances appear in the 1 H NMR spectrum (Choi et al 1996). The resonances of the first set equal those known from the ligand spectrum, whereas the other set represents the ligand bound to the insulin hexamer.…”

“…1). This tunnel and the His B10 Zn(II) ion form the anion binding site Brader et al , 1997Choi et al 1993Choi et al , 1996Brzovic et al 1994;Bloom et al 1995Bloom et al , 1997aBloom et al ,b, 1998Huang et al 1997). Two detailed studies of the structure of the Zn 2+ -R 6 hexamer in solution via NMR have been reported (Jacoby et al 1996;Chang et al 1997).…”

Crystal Structure ofallo-IleA2-Insulin, an Inactive Chiral Analogue: Implications for the Mechanism of Receptor Binding,

“…Comparison of the observed NMR spectrum with simulated spectra calculated on the basis of the crystallographic protomers reveals that the engineered monomer, although T-like in secondary structure, in part exhibits nuclear Overhauser enhancements (NOEs) between core side chains more consistent with the R-state. These observations support the view that the conformational repertoire of an insulin monomer spans the range of diverse crystal forms (29). The crystal structure of allo-Ile A2 -insulin further demonstrates that the nonstandard substitution does not perturb the surface of the protein, including side chains involved in self-association (9) and/or proposed to contact the insulin receptor (9,25,30,31).…”

Analysis of Insulin Allostery in Solution and Solid State With FTIR

“…12 Since the binding pockets for phenolic ligands are only present in the R form, a ligand independent dynamic equilibrium must form between T and R state before ligand binding. 19 Ligand binding traps the R state and shifts the equilibrium and thus the population probability towards the R state. In addition to formation of hydrophobic binding pockets the conformational changes alter the divalent cation coordination of the three His B10 for each individual trimer.…”

“…The conformational state is traditionally analysed with UV absorbing probes having a distinct binding affinity to the T 6 and R 6 state, 5,[7][8][9]19,27,28 or by near UV circular dichroism (CD) monitoring the tertiary conformational state of insulin. 3,8,10,28,29 Both techniques have several drawbacks with respect to pharmaceutical formulations including low compatibility with excipients, the need for relatively well defined conditions, and they are only applicable in solution.…”