The 27 amino acid residue polypeptide omega-conotoxin GVIA, from venom of the cone shell Conus geographus, blocks neuronal voltage-activated calcium channels at picomolar concentrations. The three-dimensional structure in aqueous solution of synthetic omega-conotoxin has been determined from two-dimensional 1H n.m.r. data recorded at 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin-spin coupling constants were used as input for distance geometry calculations with the program DSPACE. The structures were then refined using back-calculation of NOESY spectra. The family of structures obtained in this way is well defined by the n.m.r. data, the best 12 structures having pairwise root-mean-square differences of 0.68 (+/- 0.15) A over the backbone heavy atoms (N, C alpha and C) and 1.15 (+/- 0.17) A over all heavy-atoms. The molecule adopts a compact structure consisting of a small, triple-stranded, anti-parallel beta-sheet and several reverse turns. All three tyrosine residues are located on the molecular surface, which is noteworthy for its abundance of side-chain hydroxyl groups. There is no negatively charged group in conotoxin, but the five positively charged groups are distributed in three small patches on the surface, one of which, made up of the ammonium moieties of the N terminus and Lys2, may contribute to the receptor-binding surface of the molecule. An isomer of conotoxin with the same amino acid sequence, but different disulfide pairings, has also been investigated. Its structure is less well ordered than that of native conotoxin and it shows significant heterogeneity, probably as a result of cis-trans isomerism preceding hydroxyproline residues.

Amino Acid Sequence↗

PubMed1992

Synthesis and characterization of a disulfide bond isomer of omega-conotoxin GVIA.

Solid phase peptide synthesis and air oxidation of omega-conotoxin GVIA yielded, in addition to the desired product, an isomeric peptide which could be completely separated from the native toxin by repeated HPLC. A chymotrypsin-trypsin digest of this peptide, when subjected to HPLC peptide mapping, provided peptides identical with synthetic disulfide containing peptides predicted for the omega-conotoxin isomer containing C1-C2, C3-C5, C4-C6 cystinyl pairings. The 'shaking' potency (ED50 = 1500 pmoles/kg, i.c.v.) of the isomeric peptide upon cannulated rats was 1.3% of the potency of native conotoxin (ED50 = 20 pmoles/kg). Considering that all three disulfide pairings in the isomer are different from the native toxin, its retention of biological activity is of interest.

Amino Acid Sequence↗

PubMed1990

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).

Amino Acid Sequence↗

PubMed1989

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus.

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis and biological activity of six monosubstituted analogs of a sea anemone polypeptide neurotoxin.

Monosubstituted analogs of Stichodactyla helianthus neurotoxin I (ShI), a sea anemone polypeptide, were synthesized by solid-phase methods. The analogs were selected to probe the importance of charged residues in the amino-terminal region; ionizable side chains were replaced with a corresponding neutral isosteric amino acid side chain. Following oxidation of the three disulfide bonds and refolding, the analogs were purified by gel, ion-exchange and reversed-phase chromatography. Circular dichroism and fluorescence spectral analyses indicate that the analogs have folded structures very similar to the native toxin. Crustacean paralytic bioassays and neuronal receptor binding assays of ShI analogs show that the anionic triad Asp 6-Asp 7-Glu 8 is essential for activity, as replacement of any of these side chains with the corresponding amide reduces toxicity and binding at least 1000-fold. Substitution at Lys 4 or Asp 11 reduces biological activity at least 10-fold; these residues are apparently not as critical as the polyanionic region. Replacing the N-terminal Ala residue with a Tyr residue has only a slight effect upon crab toxicity and axolemma binding. Our results suggest that several amino acid side chains near the N-terminus of this sea anemone neurotoxin are important for binding to crab neuronal sodium channels.

Amino Acid Sequence↗

PubMedDate not supplied

Evaluation of TiCl4-mediated reduction of methionine sulfoxide in peptides with oxidizable or reducible residues.

Reduction of methionine sulfoxide with TiCl4/NaI is very rapid for simple methionine-containing peptides. The utility of this oxido/reduction system has been evaluated for three model peptides that contain oxidation/reduction-sensitive components such as a disulfide bond and/or a tryptophan residue. Completely specific reduction of methionine sulfoxide without some reduction of the disulfide bond was not possible with TiCl4/NaI. Reduction of the methionine sulfoxide residue in these model peptides yielded the desired product as the major component (yield ca. 70%) when a reaction time of four minutes was used. Methionine sulfoxide appears to be the most readily reducible species by low valent titanium. The competing side reactions observed were disulfide bond reduction by low valent titanium and/or tryptophan oxidation by the I2 generated by reduction of the TiCl4 with NaI. These side reactions became a serious problem when longer reaction times were used. The levels of contaminants generated by these side reactions were observed to increase with time, reducing the yield of the desired product.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.

Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of the molecule. Upon cleavage of DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (DABCYL-ICE-EDANS), an increase in fluorescence is observed at the EDANS emission wavelength of 490 nm, permitting a continuous assay of ICE that is useful in the screening of inhibitory compounds. The Km and kcat results for hydrolysis of DABCYL-ICE-EDANS by ICE were 11.4 +/- 1.6 microM and 0.79 +/- 0.4 s-1. The second order rate constant for hydrolysis of this substrate (kcat/Km = 7.0 +/- 1.3 x 10(4) M-1 s-1) is comparable to that for the cleavage of the previously described fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-AMC (6.4 x 10(4) M-1 s-1).

Amino Acid Sequence↗