A Proton-Magnetic-Resonance Study on the Molecular Conformation and Structure- Function Relationship of a Long Neurotoxin, Laticauda Semifasciate III fromLaticauda semifasciata

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Abstract
The 300- and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from L. semifasciata were analyzed. Comparison with the NMR spectra of .alpha.-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to LS III, allowed the assignment of all aromatic proton resonances to specific amino acid residues. All methyl proton resonances were assigned to specific types of amino acid residues. The pH dependences of the aromatic and methyl proton chemical shifts were analyzed by the non-linear least-square method to give the pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured to elucidate the spatial proximity of the methyl-bearing residues and aromatic residues. On the basis of these NMR data and using the crystal structure of .alpha.-cobratoxin by Walkinshaw more than half of the methyl proton resonances were assigned to specific amino acid residues. A hydrophobic core comprising the 1st loop, the central loop and the tail part of the molecule was defined. This hydrophobic core may be common to all long neurotoxins and may protect the 3-stranded antiparallel pleated .beta.-sheet structure, making the backbone structure of long neurotoxins more rigid than that of short neurotoxins. The positively charged surface of LS III, which was responsible for binding to the acetylcholine receptor protein, was confirmed as the concave surface formed by the central and the 3rd loop. The arrangement of the amino acid residues on this surface was similar to that of all other neurotoxins. The slow on-off rates of association of long neurotoxins with receptor arose from the rigid backbone structure. A small conformation change was probably associated with binding to the receptor protein.