Molecular and Cellular Biochemistry86:87-95 (1989) © 1989 KluwerAcademic Publishers - Printed in the Netherlands 87 Original Article Molecular topography and secondary structure comparisons of botulinum neurotoxin types A, B and E Bal Ram Singh and B. R. DasGupta Food Research Institute, 1925 Willow Drive, University of Wisconsin, Madison, WI 53706, USA Received 16 May 1988; accepted 5 October 1988 Key words~" botnlinum neurotoxin, derivative spectra, tyrosine exposure, secondary structure, circular dichro- ism Abstract Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (a-helix,/3-sheet, ~turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantmn yield). The proteins are high in 13-pleated sheet content (41-44070) and low in a-helical content (21-28%). About 30-36070 of the amino acids are in random coils. The/3-sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and E NT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free trypto- phan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low a-helix and high 13-sheets) and tertiary (exposed tyrosine residues and trypto- phan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities. Introduction Botulinum neurotoxins (NT), a group of serological- ly distinct proteins (- 150 kDa) produced by differ- ent strains of Clostridium botulinum, are responsi- ble for the neuroparalytic disease botulism. The NT binds to the neuromuscular junctions, enters the nerve ceils, blocks the release of a neurotrausmitter, acetylcholine, thus causes flaccid paralysis. The sev- en serotypes are known as NT types A through G [1-31. The single chain polypeptide (~ 150 kDa) under- goes a post synthetic cleavage (nicking) by protease endogenous to the bacteria in some eases (e.g. type A) but not in others (e.g. type E). The nicked (i~ dichain) NT has two chains (light and heavy of - 50 and - 100 kDa, respectively) linked through at least one disulfide bond. The NT is therefore recovered from the bacterial cultures as single chain (type E), dichain (type A) or a mixture of single and dichain (type B) molecules. The single chain NTs can be nicked by trypsin into dichain forms [4]. The rela- tively low toxicity (LDs0/mg protein) of newly syn- thesized NT is enhanced (activated) in the bacterial culture by endogenous protease(s) identity of which is not clear [5]. When the bacterial culture lacks the activation protease(s) e.g. type E, the NT remains unactivated which can be activated 100-fold or more