NMR analysis and sequence of toxin II from the sea anemone Radianthus paumotensis

Abstract

Toxin II from Radianthus paumotensis (RpII) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of beta-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular alpha-helical segments. The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.

Linear and cyclic peptide analogues of the polypeptide cardiac stimulant, anthopleurin‐A

“…8) has been noted previously and attributed to secondary or tertiary folding interactions [5,37l. The data for the type 2 polypeptides Stichodactyla helianthus neurotoxin I [38] and Heteractispaumotensis toxins I1 and I11 [39,40] show that the homologous protons also have perturbed chemical shifts. The presence of such a shift in the peptides as well as in the intact proteins indicates that the interaction causing the perturbation must be short-range rather than long-range.…”

Functional discrimination of sea anemone neurotoxins using 3D-plotting

“…The corresponding three-dimensional representation in Figure 4 results in The sequences were obtained from the following papers: AeI (Actinia equina): [27], AETX I (Anemonia erythraea): [28], APE 1-1, APE 1-2, APE 2-1 (Anthopleura elegantissima): [29], AFT-I, AFT-II (Anthopleura fuscoviridis): [30], ATX-Ia, ATX-I, ATX-II, ATX-V ATX-III, ATX-IV (Anemonia sulcata): [31][32][33][34][35][36], Ap-A, Ap-B, PCR1-2, PCR 2-1, PCR2-5, PCR 2-10, PCR 3-3, PCR 3-6, PCR 3-7 (Anthopleura xanthogrammica): [37][38][39], BcIII (Bunodosoma caissarum): [40], Bg II, Bg III (Bunodosoma granulifera): [41], Cp II (Condylactis passiflora): [42], RTX-I, RTX-II, RTX-III, RTX-IV, RTX-V (Radianthus macrodactylus/previously Heteractis macrodactylus): [43][44][45], RpII, RpIII (Radianthus paumotensis/previously Heteractis magnífica or Heteractis paumotensis): [46][47], Sh I (Stichodactylae helianthus): [48], CLX-I, CLX-II (Calliactis parasitica): [19], Halcurin (Halcurias carlgreni): [49], PA-TX (Parasicyonis actinostoloides/previously Entacmaea quadricolor): [50].…”

The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis

“…However, in Rpm the critical residue is replaced by Glu ( fig.3). It will be of interest to compare the three-dimensional structure of this region of Rpm with that of RpII [2]. a Comparisons were made by the ALIGN program and are expressed as alignment scores.…”

“…An alignment score of greater than 3 (standard deviations) corresponds to a probability of less than 0.0014 that a randomly generated sequence of-the same composition would produce a better score. Toxins Rp are from Radianthus paumotensis [2], As are from Anemonia sulcata [4][5][6] and Ax is from Anthopleura xanthogrammica [3] (cf. fig.3) …”

“…Its amino acid sequence is homologous with toxins of two other sea anemones, Anthopleura xanthogramrnica [31 and Anemonia sulcata [4- Pooled peptides V8-1, V8-3, V8-5 and V8-7 were analyzed in detail ( fig.l). The composition of the fraction eluting at 20 min indicated that it contained a small amount of a peptide comprising residues 9-28. reduced and carboxymethylated as described [2]. Amino acid compositions were determined with a Waters Picotag system [7] or with a Dionex D-500 analyzer.…”