Raman signatures of ligand binding and allosteric conformation change in hexameric insulin

Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T, T,R,, and R,). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of phenolic ligands and the binding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenzoic acid (4N3N) to the allosteric sites of T3R3 and R-6. The RD spectroscopic studies show changes in the amide I and III bands for the transition of residues B-1-B-8 from a meandering coil to an a helix in the T-R transitions and identify the Raman signatures of the structural differences among the T, TR,, and R-6 states. Evidence of the altered environment caused by the similar to 30 Angstrom displacement of phenylalanine (Phe) B1 is clearly seen from changes in the Raman bands of the Phe ring. Raman signatures arising from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) sites show these carboxylates give distorted, asymmetric coordination to Zn(II). The RD spectra also reveal the importance of the position and the type of substituents for designing aromatic carboxylates with high affinity for the His B10 metal site. (C) 2001 John Wiley & Sons, Inc.