Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone.
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.
Monks
S A
SA
School of Biochemistry, University of New South Wales, Kensington, Australia.
Gould
A R
AR
Lumley
P E
PE
Alewood
P F
PF
Kem
W R
WR
Goss
N H
NH
Norton
R S
RS
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003064:Cnidarian Venoms / Q000737:chemistry*; D004789:Enzyme Activation; D008239:Lysine / Q000737:chemistry; D008666:Metalloendopeptidases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry*; D012997:Solvents; D013329:Structure-Activity Relationship; D014357:Trypsin
D003064:Cnidarian Venoms; D009498:Neurotoxins; D012997:Solvents; C059713:neurotoxin I, Stichodactyla helianthus; D001120:Arginine; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D008239:Lysine