Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?

Botulinum neurotoxin, produced by Clostridium botulinum as a similar to 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The similar to 50- and similar to 100-kDa chains of the di-chain molecule remain held together by an interchain disulfide bridge and noncovalent interactions, The neurotoxin hinds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the similar to 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state, The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing, The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and Das-Gupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-630), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel. (C) 1999 Academic Press.