Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone

Stephen A. Monks, Alison R. Gould, Paul E. Lumley, Paul F. Alewood, William R. Kem, Neil H. Goss, Raymond S. Norton

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

Abstract

The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.

Original languageEnglish
Pages (from-to)93-101
Number of pages9
JournalBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Volume1207
Issue number1
DOIs
Publication statusPublished - 20 Jul 1994
Externally publishedYes

Keywords

  • (Sea anemone)
  • Neurotoxin
  • Proteolysis
  • Solvent accessibility
  • Structure-function relationship

Cite this