title: Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus.
abstract: Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA. blumenkm@uc.edu
authors.forename: P H
authors.initials: PH
authors.lastname: Wen
authors.forename: K M
authors.initials: KM
authors.lastname: Blumenthal
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D008387:Marine Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D008387:Marine Toxins; D009498:Neurotoxins