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

Hexameric insulin is an allosteric protein that undergoes transitions betwe en three conformational states (T, T,R,, and R,). These allosteric states a re 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) sp ectroscopy is utilized to examine the binding of phenolic ligands and the b inding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenz oic acid (4N3N) to the allosteric sites of T3R3 and R-6. The RD spectroscop ic 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 transition s 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 se en from changes in the Raman bands of the Phe ring. Raman signatures arisin g from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) s ites 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.