Evidence for conformational differences in aqueous and crystalline structures of α-bungarotoxin and cobratoxin

Abstract

Laser Raman spectroscopy has been employed to investigate the structures of α-bungarotoxin ( Bungarus multicinctus) and cobratoxin ( Naja naja siamensis) in H 2O and D 2O solutions. Structures of the aqueous neurotoxins are compared with one another and with the X-ray crystal structures. The results indicate that the solution and crystal molecular structures of cobratoxin are in substantial agreement with one another, but those of α-bungarotoxin are not. Raman data provide no evidence for strained disulfides in aqueous α-bungarotoxin, although strained CSSC dihedral angles are indicated for the X-ray crystal structure. The data are interpreted as evidence for a strained molecular conformation of α-bungarotoxin in the crystal, which converts to a relaxed, more energetically favorable conformation in aqueous solution. Raman spectra also suggest more β-strand secondary structure in aqueous α-bungarotoxin (47 ± 5%) than in the crystalline form ( < 10%). The high β-strand content measured by Raman spectroscopy could be due to either a secondary structure in solution that is appreciably different than that of the crystal, or to the imprecision of the Raman method in distinguishing peptide configurations that are vibrationally equivalent but conformationally inequivalent. Aqueous α-bungarotoxin and cobratoxin also differ from one another in amino acid side chain orientations and interactions, though not in main chain conformations. Different geometries are indicated for cystine CCSS dihedral angles, and different hydrogen bonding states are indicated for internal tyrosines. Tyrosine-24 of α-bungarotoxin is shown to donate a strong hydrogen bond to a negative acceptor, deduced to be glutamate-41, whereas the equivalently positioned residue of cobratoxin is apparently hydrogen bonded to solvent molecules.