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Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: calculation of tertiary structure using systematic homologous model building, dynamical simulated annealing, and restrained molecular dynamics

Sutcliffe, M.J.; Dobson, C.M.; Oswald, R.E.

Biochemistry 31(11): 2962-2970

1992


ISSN/ISBN: 0006-2960
PMID: 1550821
DOI: 10.1021/bi00126a017
Accession: 007803181

Neuronal bungarotoxin has previously been shown, using two-dimensional 1H NMR spectroscopy, to have a triple-stranded antiparallel .beta.-sheet structure which dimerizes in solution [Oswald, R. E., Sutcliffe, M. J., Bamberger, M., Loring, R. H., Braswell E., and Dobson, C. M. (1991) Biochemistry 30, 4901-4909]. In this paper, structural calculations are described which use the 582 experimentally measured NOE restraints in conjunction with 27 .phi. angle restraints from J-value measurements. The positions of the N-terminal region and C-terminal region were poorly defined in the calculated structures with respect to the remainder of the structure. The region of the structure containing the triple-stranded .beta.-sheet was, however, well defined and similar to that found in the structure of homologous .alpha.-bungarotoxin (45% amino acid identity. The experimental restraints did not result in a well-defined dimer interface region because of the small number of NOEs which could be identifed in this region. An approach was therefore adopted which produced model structures based to varying degrees on the .alpha.-bungarotoxin structure. Fourteen different structures were generated in this manner and subsequently used as starting points for refinement using dynamical simulated annealing followed by restrained molecular dynamics. This approach, which combines NMR data and homologous model building, has enabled a family of structures to be proposed for the dimeric molecule. In particular, Phe 49 has been identified as possibly playing an important role in dimer formation, this residue in one chain interacting with the corresponding residue in the adjacent chain.

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