title: Mutagenesis of Cerebratulus lacteus neurotoxin B-IV identifies NH2-terminal sequences important for biological activity.
abstract: We have previously demonstrated that a synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV can be expressed in bacteria, and the recombinant toxin purified and refolded (Howell, M. L., and Blumenthal, K. M. (1989) J. Biol. Chem. 264, 15268-15273). This toxin, which contains an NH2-terminal methionine residue not present in authentic B-IV, has a specific toxicity 35-40% that of the naturally occurring form. Deletion of the codon for the NH2-terminal methionine allows expression of fully active recombinant B-IV, demonstrating that hydroxylation of Pro-10 is not important for biological activity. Site-directed mutagenesis of the des-Met(-1) form has been employed to analyze the contribution of NH2-terminal sequences of this toxin to its activity. We have emphasized replacement of helix-favoring residues by helix-destabilizing ones which are otherwise sterically similar. When Ala-3 or Ala-8 is replaced by serine, little or no effect on specific toxicity is observed. However, the double mutant in which both alanines are substituted with serine is more than twice as active as natural B-IV, although the secondary structures and conformational stabilities of the wild-type and mutant forms are the same. When Ala-3 and 8 are simultaneously replaced with glycine, the resulting toxin displays an activity similar to that of the wild-type form. The conformational properties of this mutant are unchanged from that of either wild-type or the serine double mutant. These data indicate that insertion into the NH2-terminal region of toxin B-IV of residues which can participate in hydrogen bond formation enhances biological activity of the protein.
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
authors.forename: M L
authors.initials: ML
authors.lastname: Howell
authors.forename: K M
authors.initials: KM
authors.lastname: Blumenthal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea; D001483:Base Sequence; D001681:Biological Assay; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008387:Marine Toxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000235:genetics*; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000633:toxicity
chemical_list: C028056:Cerebratulus lacteus toxin B-IV; D008387:Marine Toxins; D009498:Neurotoxins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins