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The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).

The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).

To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the alpha-cobratoxin-alpha7 receptor complex. This model was achieved by using (i) a three-dimensional model of the alpha7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the alpha-cobratoxin and alpha7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin-receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists.

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.

Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra- and intermolecule cross-linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross-linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp(29) of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross-linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde-modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme-linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp(29) of cobrotoxin and glutaraldehyde-modified derivatives was formed differently. These results suggest that glutaraldehyde cross-linking leads to a change in the microenvironment of cobrotoxin Trp(29) and alters the pathway of its fine structure formation during the refolding of cobrotoxin.

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.

We have solved the crystal structure of aphrodisin, a pheromonal protein inducing a copulatory behaviour in male hamster, using MAD methods with selenium, at 1.63 A resolution. The monomeric protein belongs to the lipocalin family, and possesses a disulfide bridge in a loop between strands 2 and 3. This disulfide bridge is characteristic of a family of lipocalins mainly identified in rodents, and is analogous to the fifth disulfide bridge of the long neurotoxins, such as alpha cobratoxin. An elongated electron density was found inside the buried cavity, which might represent a serendipitous ligand of unknown origin. The analysis of the water accessible surfaces of the side-chains bordering the cavity indicates that Phe76 may be the door for the natural ligand to access the cavity. This residue defines the entry of the cavity as belonging to the consensus for lipocalins. The face bearing Phe76 might also serve for the interaction with the receptor., (Copyright 2001 Academic Press.)

The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.

Several investigators have isolated nerve growth factor (NGF) from various tissues and organs of different animals. There is no published documentation about NGF from body fluids, such as blood serum, saliva, and urine. Contrary to the unsuccessful attempts to detect or isolate NGF in serum in the past, this investigation reports the isolation of NGF from human serum, saliva, and urine. It further reports the comparison of properties between NGFs derived from cobra venom and cobra serum. NGF from serum, saliva, and urine was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The identified fractions of NGF were further purified to study the biological and immunological properties. The biological activities of NGFs from human body fluids and cobra serum on PC12 cells were miniscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from human serum, saliva, and urine were identical, 36.0 kDa. However, the molecular weights of cobra serum and cobra venom NGFs were different, 55.0 kDa and 13.5 kDa, respectively. NGF level is age dependent and varies under different conditions. Using anti-human NGF, diagnostic tests can be developed for neurological disorders. This investigation also emphasizes the replacement of invasive blood collection for serum by use of saliva and urine for clinical diagnostic use in general.

Long chain curarimimetic toxins from snake venom bind with high affinities to both muscular type nicotinic acetylcholine receptors (AChRs) (K(d) in the pm range) and neuronal alpha 7-AChRs (K(d) in the nm range). To understand the molecular basis of this dual function, we submitted alpha-cobratoxin (alpha-Cbtx), a typical long chain curarimimetic toxin, to an extensive mutational analysis. By exploring 36 toxin mutants, we found that Trp-25, Asp-27, Phe-29, Arg-33, Arg-36, and Phe-65 are involved in binding to both neuronal and Torpedo (Antil, S., Servent, D., and Ménez, A. (1999) J. Biol. Chem. 274, 34851-34858) AChRs and that some of them (Trp-25, Asp-27, and Arg-33) have similar binding energy contributions for the two receptors. In contrast, Ala-28, Lys-35, and Cys-26-Cys-30 selectively bind to the alpha 7-AChR, whereas Lys-23 and Lys-49 bind solely to the Torpedo AChR. Therefore, alpha-Cbtx binds to two AChR subtypes using both common and specific residues. Double mutant cycle analyses suggested that Arg-33 in alpha-Cbtx is close to Tyr-187 and Pro-193 in the alpha 7 receptor. Since Arg-33 of another curarimimetic toxin is close to the homologous alpha Tyr-190 of the muscular receptor (Ackermann, E. J., Ang, E. T. H., Kanter, J. R., Tsigelny, I., and Taylor, P. (1998) J. Biol. Chem. 273, 10958-10964), toxin binding probably occurs in homologous regions of neuronal and muscular AChRs. However, no coupling was seen between alpha-Cbtx Arg-33 and alpha 7 receptor Trp-54, Leu-118, and Asp-163, in contrast to what was observed in a homologous situation involving another toxin and a muscular receptor (Osaka, H., Malany, S., Molles, B. E., Sine, S. M., and Taylor, P. (2000) J. Biol. Chem. 275, 5478-5484). Therefore, although occurring in homologous regions, the detailed modes of toxin binding to alpha 7 and muscular receptors are likely to be different. These data offer a molecular basis for the design of toxins with predetermined specificities for various members of the AChR family.

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.

We report the amino acid sequence of a basic protein isolated from the snake venom of Naja naja atra. An automated Edman sequencer was used to determine the 65-residue sequence, aided by electrospray ionization/mass spectrometry. Online reduction and pyridylethylation of the peptide was performed to identify the cysteine residues. Trypsin, chymotrypsin and aspartic digestions were carried out to derive peptide fragments for further sequencing. Fragmented peptides were overlapped to obtain the complete sequence. Molecular mass measurements of the whole protein and its fragments were used as a countercheck for sequence assignment. Further confirmation of the sequence was indicated by sequence homology to other snake venom neurotoxins. A molecular model of the tertiary structure was constructed based on sequence homology, and was refined by global minimization and extensive quality control algorithms. Electrostatic and hydrophobic surface calculations and molecular dynamics simulations were carried out to determine the functional properties of the molecule.

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones. Loop I, half of loop II and the C-terminal residue of fasciculin 2 were therefore transferred into the toxin alpha. The inhibition constant of the resulting chimera is only 15-fold lower than that of fasciculin 2, and as expected the potency of binding to the toxin alpha target has been lost. In order to understand the structure-function relationship between the chimera and its "parent" molecules, we solved its structure by X-ray crystallography. The protein crystallized in space group P3(1)21 with a=b=58.5 A, and c=62.3 A. The crystal structure was solved by molecular replacement and refined to 2.1 A resolution. The structure belongs to the three-fingered snake toxin family with a core of four disulphide bridges from which emerge the three loops I, II and III. Superimposition of the chimera on fasciculin 2 or toxin alpha revealed an overall fold intermediate between those of the two parent molecules. The regions corresponding to toxin alpha and to fasciculin 2 retained their respective geometries. In addition, the chimera protein displayed a structural behaviour similar to that of fasciculin 2, i.e. dimerization in the crystal structure of fasciculin 2, and the geometry of the region that binds to acetylcholinesterase. In conclusion, this structure shows that the chimera retains the general structural characteristics of three-fingered toxins, and the structural specificity of the transferred function., (Copyright 2000 Academic Press.)

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule., (Copyright 1999 Academic Press.)

The review is mainly devoted to snake venom alpha-neurotoxins which target different muscle-type and neuronal nicotinic acetylcholine receptors. The primary and spatial structures of other snake venom proteins as well as mammalian proteins of the Ly-6 family, which structurally resemble the 'three-finger' snake proteins, are also briefly discussed. The main emphasis is placed on recent data characterizing the alpha-neurotoxin interactions with nicotinic acetylcholine receptors.

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding., (Copyright 1999 Academic Press.)

Cobrotoxin is the main neurotoxic protein isolated from the venom of Taiwan cobra Naja naja atra. It is a small, basic protein consisting of a single polypeptide chain of 62 amino acids, cross-linked by four disulfide bonds. The disulfide bonds and Tyr-25 which is buried in the molecule form a central core to maintain and stabilize the active conformation of the toxin. Selective and stepwise chemical modifications of cobrotoxin indicate that at least two cationic groups, an epsilon-amino group of Lys-47 and a guanidino group of Arg-33, both of which are common to all known postsynaptic neurotoxins, held at a certain critical distance in the molecule, are functionally important for its neuromuscular blocking activity. The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription polymerase chain reaction. Sequencing several clones containing about 0.5 Kb DNA inserts contained a complete and full-length reading frame of 249 base pairs covering a precursor of cobrotoxin gene with a deduced mature protein sequence of 62 amino acids which are identical to the amino acid sequence of cobrotoxin and a 21 amino acid segment of signal peptide. Expression of cobrotoxin in E. coli vector generated a polypeptide which can cross-react with the antisera against the native cobrotoxin.

Motions of the backbone C alpha H alpha and threonine C beta H beta bonds of toxin alpha were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H-->13C NOE were obtained, as well as the variations of R1 rho (90 degrees) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several C alpha H alpha and threonine C beta H beta experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin alpha, a highly stable protein (Tm = 75 degrees C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 microseconds to 10 ms.

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.

Cardiotoxin and neurotoxin analogues isolated from snake venom sources are highly homologous proteins (>50% homology) with similar three-dimensional structures but exhibit drastically different biological properties. In the present study, we compare the conformational stability of cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX), a neurotoxin analogue, from the Taiwan cobra (Naja naja atra), using circular dichroism spectroscopy and hydrogen-deuterium (H/D) exchange techniques in conjunction with two-dimensional NMR methods. Contrary to expectations, it is found that CTX III and CBTX differ significantly in their structural stabilities. The three-dimensional structure of CBTX is less stable than that of CTX III. The amide protons of residues at the N- and C-terminal ends of the CTX III molecule are strongly protected against H/D exchange, implying that the terminal ends of the molecule are bridged together by significant numbers of hydrogen bonds. However, in CBTX, amide protons at the terminal ends of the molecule do not exhibit an significant protection against H/D exchange. Comparison of the protection factors of the various amide protons in CTX III and CBTX reveals that the extraordinary stability of CTX III stems from the strong network of interactions among the residues at the N- and C-terminal ends and also due to the tight and ordered packing of the nonpolar residues involved in the triple-stranded, anti-parallel, beta-sheet segment of the molecule., (Copyright 1999 Academic Press.)

A 600 MHz NMR study of alpha-conotoxin ImI from Conus imperialis, targeting the alpha7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin-spin coupling constants, and amide protons hydrogen-deuterium exchange data: rmsd of the backbone atom coordinates at the 2-12 region is 0.28 A in the 20 best structures. The structure is described as a type I beta-turn (positions 2-5) followed by a distorted helix (positions 5-11). Similar structural patterns can be found in all neuronal-specific alpha-conotoxins. Highly mobile side chains of the Asp-5, Arg-7 and Trp-10 residues form a single site for ImI binding to the alpha7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.

Toxic proteins are produced by a diversity of venomous animals from various phyla. They are often of small size, possess a large density of disulfide bonds and exert multiple functions directed toward a variety of molecular targets, including a diversity of enzymes and ion channels. The aim of this brief and non-exhaustive review is three-fold. First, the structural context associated with the functional diversity of animal toxins is presented. Among various situations, it is shown that toxins with a similar fold can exert different functions and that toxins with unrelated folds can exert similar functions. Second, the functional sites of some animal toxins are presented. Their comparison shed light on how (i) distinct functions can be exerted by similarly folded toxins and (ii) similar functions can be shared by structurally distinct toxins. Third, it is shown that part of the functional site of foreign proteins can be grafted on an animal toxin scaffold, opening new perspectives in the domain of protein engineering.

A disulfide isomerization at the C-terminus of cobrotoxin occurred spontaneously by dissolving in alkali buffer. Irreversible conversion of cobrotoxin into its isomers was completely achieved within 4 days. The isomerization reaction was decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic, and GSSG was the most effective one among the thiol compounds used. Moreover, the oxidized thiol compounds were always superior to reduced ones in decreasing the rate of disulfide interchange. To further assess the intrinsic elements essential for the occurrence of disulfide isomerization of cobrotoxin, the toxin molecule was subjected to modification on its Arg, Lys, Trp, Tyr, and carboxyl groups. In sharp contrast to other modified derivatives, the isomerization reaction was decelerated by trinitrophenylation on Lys-26, Lys-27, and Lys-47, whereas it was rapidly completed after modification of carboxyl groups. Neither chemical modification nor the toxin's conformation affected the irreversibility of isomerization reaction. Thus, the observed change in the rate of disulfide isomerization reflects the involvement of Lys residues and carboxyl groups in this reaction. Although thiol compounds further decelerated the conversion of trinitrophenylated cobrotoxin into its isomers, they did not exert a notable effect on the isomerization of carboxyl groups-modified derivative. These results clearly indicate that disulfide isomerization of cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule, and that the thiol compounds are coordinated with the negatively charged groups of cobrotoxin to exert their inhibitory action., (Copyright 1998 Academic Press.)

Refolding of cobrotoxin was assessed by the exposure degree of its single Trp determined by an acrylamide quenching study. The change in the accessibility of Trp for acrylamide quantitatively reflected the formation of folded cobrotoxin, and the data were confirmed by HPLC and gel electrophoresis analyses. However, the site-specific information provided by quenching Trp fluorescence revealed that the ordered structure in the neighborhood of Trp was attained prior to the complete formation of the tertiary structure of cobrotoxin. HPLC analyses showed that, in addition to refolded cobrotoxin, two novel species (cobrotoxin II and cobrotoxin III) with isomerization of disulfide bonds at the C-terminus of the toxin molecule were produced along the folding reaction. The disulfide pairings in cobrotoxin II and cobrotoxin III were Cys43-Cys55 and Cys54-Cys60 and Cys43-Cys60 and Cys54-Cys55, respectively. Among the three possible two-disulfide species at the C-terminus, the disulfide linkages Cys43-Cys60 and Cys54-Cys55 of cobrotoxin III caused a marked decrease in lethality and resulted in a conformation which was notably different from that observed with the native toxin molecule as evidenced by CD spectra. The refolding reaction was accelerated by the addition of GSH/GSSG, and the resulting products were mostly folded cobrotoxin. However, if GSH/GSSG was not added into the initial folding materials, the yields of cobrotoxin II and cobrotoxin III greatly increased. The conversion of cobrotoxin to its isomers was to be irreversible and pH-dependent: the higher the pH, the faster the rate of conversion. However, this conversion could be partly inhibited by GSH/GSSG. Cobrotoxin II and cobrotoxin III were purified from Taiwan cobra venom as well, and their yields in comparison to that of cobrotoxin in venom were similar to that noted with the folded products in the presence of GSH/GSSG. Moreover, the rate of disulfide isomerization was expected to be slow in venom fluid in which the pH was approximately pH 6.2. Thus, the finding that cobrotoxin represents the predominant neurotoxin species in Taiwan cobra venom is probably associated with the synergistic effects of GSH/GSSG and pH.

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.

Two novel postsynaptic neurotoxins (alpha-neurotoxins) isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct primary sequences and different neurotoxicities as compared with the most abundant and lethal component in the venom, i.e., cobrotoxin characterized before from the same venom. The complete sequences of two neurotoxin analogues were determined by N-terminal Edman degradation and comparison of amino acid compositions of proteolytic toxin fragments with other homologous toxins of known sequences. The short-chain neurotoxin consists of 61 amino acid residues with eight conserved cysteine residues and is found to show 78% sequence identity with cobrotoxin. The other toxin, consisting of 65 residues with ten cysteines, belongs to the family of long-chain neurotoxins. It is the first long-chain alpha-neurotoxin reported from the Taiwan cobra. The lethal toxicities of these two novel neurotoxins were much lower than cobrotoxin, albeit with close structural homology among the three toxins in terms of their primary sequences and tertiary structure predicted by homology modeling. Multiple sequence alignment and comparison coupled with construction of a phylogenetic tree for various alpha-neurotoxins of Naja and closely related genuses have established that all nicotinic alpha-neurotoxins present in the snake family of Elapidae are closely related to each other, presumably derived from an ancestral polypeptide by gene duplication and subsequent multiple mutational substitutions.

Although few proteins have been studies as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C11 and C502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C11-->F and C502-->T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacement reduced it to (25 +/- 2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C11 and C502 in the native CSA that joins the chain ends to form a dense conformation.

Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein. Analysis of these NMR measurements demonstrates that toxin alpha exhibits various time scale motions, i.e., particularly large amplitude picosecond to nanosecond motions at the tips of the loops, observable microsecond to millisecond motions around two disulfide bridges, second time scale motions around the C-N bonds of asparagine and glutamine side chains which are more or less rapid depending on their amino acid solvent accessibility, and minute to hour motions in the beta-sheet structure. The less well-defined regions of toxin alpha solution structures are subject to important picosecond to nanosecond motions. The toxic site is organized around residues belonging to the rigid core of the molecule but also comprises residues exhibiting dynamics on various time scales. The Malpha1 epitope is subject to large picosecond to millisecond motions, which are probably modified by the interaction with the antibody. This phenomenon could be linked to the neutralizing properties of the antibody.

A novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2,386bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.

Two genomic DNAs with the size of 2.3 kb and 2.4 kb, which were isolated from the liver of Naja naja atra (Taiwan cobra), encoded the precursors of cardiotoxin 4 and cobrotoxin, respectively. Both genes shared virtually identical overall organization with three exons separated by two introns, which were inserted in the similar positions of the gene's coding regions. Moreover, their nucleotide sequences shared approximately 84.2% identity. This result reveals the evolutionary relationship between cardiotoxin and cobrotoxin. The exon/intron structures of cardiotoxin 4 and cobrotoxin genes were similar to that reported for erabutoxin c gene, a neurotoxin genomic DNA from a sea snake (Laticauda semifasciata). However, in contrast to the finding that the intron 2 of these genes had a similar size, a notable variation with the size of intron 1 was observed (1233 bp, 1269 bp and 197 bp for cardiotoxin 4, cobrotoxin and erabutoxin c genes, respectively). The different size with intron 1 is due to the middle region at the first intron of cardiotoxin 4 and cobrotoxin genes, which encoded small nucleolar RNA (snoRNA), being absent in that of erabutoxin c gene. These results, together with the finding of the potential mobility of snoRNA genes during evolution, suggest that intron insertions or deletions of snoRNA genes occur with the evolutionary divergence of snake neurotoxins and cardiotoxins.

A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC. The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase. This novel toxin comprises 72 amino acid residues with 10 cysteines. The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom. The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively. Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin. Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69. A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified. The neurotoxicity was further decreased when Arg-9 was additionally modified. These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR). The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.

A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified alpha-toxin from Naja nigricollis to a resin. The alpha-toxin was monothiolated on the epsilon-amino group of its lysine-15 by reaction with N-succinimidly-3-(2-pyridyldithio)propionate and was covalently linked in a reversible manner to a thiopropyl-activated agarose resin by thiol-disulfide exchange. We found that 50% of the immobilized toxin molecules were effective for purifying nAChR, indicating a high accessibility of resin-bound toxins to their binding sites on the receptor protein. Purified alpha-toxin/nAChR complexes were eluted with nearly 100% recovery by reduction of disulfide bridges with dithiothreitol. nAChR solutions of high purity were obtained, as shown by polyacrylamide gel electrophoresis. A comparison was made with two other procedures of affinity chromatography using: (1) alpha-bungarotoxin from Bungarus multicinctus polymodified on several amines and covalently linked to a resin in a reversible manner, and (2) a commercial agarose resin bearing irreversibly immobilized alpha-cobrotoxin from Naja naja kaouthia. We conclude that: (1) the use of a selected regioselective linking of a peptidic ligand to a chromatography resin results in an increased efficiency of protein binding, and (2) a high yield of protein recovery is obtained via reversible covalent linking.

Retro-inversion is considered an attractive approach for drug and vaccine design since it provides the modified peptides with higher resistance to proteolytic degradation. We therefore investigated in detail the effect of retro-inversion on the immunological properties of synthetic peptides. We have synthesized retro-inverso analogues of MHC II restricted peptides that thus contained the correct orientation of the side chains but an inverse main chain. Retro-inversion made the peptides unable to compete in I E(d) or I A(d) binding tests, demonstrating a very low, if any, capacity to bind to MHC II molecules. These results confirm previous structural data that hydrogen bonds between residues of MHC II molecules and the main chain of antigenic peptides play a major interacting role. In vito experiments further showed that retro-inversion of a T-cell epitope causes its inability to either sustain in vitro T-cell stimulation or to prime specific T cells. Moreover, the retro-inverso peptide was not recognized by antibodies raised against the native peptide and did not elicit antibodies when injected into BALB/c mice. Retro-inverso peptides appear to be poor immunogens as a result of their weak capacity to bind to MHC II molecules. As an advantage, they are not expected to trigger undesirable humoral responses such as hypersensitivity or allergic disease. These results also provide a molecular explanation regarding the weak immunogenicity of D-amino acids containing polypeptides.

The effect of 2,2,2-Trifluoroethanol (TFE) on the structure of five all beta-sheet proteins, isolated from the venom of the Taiwan cobra (Naja naja atra), is studied. In all the toxins used, it is observed that significant amount of alpha-helix is induced at higher concentrations of TFE. In all these proteins, the induction of helical conformation and disruption of the tertiary structure seem to occur simultaneously. The structural transitions induced by TFE in reduced and denatured protein appear to be different from those observed in the native protein(s). In our opinion, the findings reported herein could have significant implications on research in the area of protein folding.

In order to identify the structural determinants of cobrotoxin that caused its anomalous mobility in sodium dodecyl sulfate (SDS) gel, comparative studies on the capacities of cobrotoxin and its modified derivatives for binding with SDS were made. Cobrotoxin migrates faster in SDS gel after modification of Lys residues or carboxylated groups. However, the capacities of modified derivatives for binding with SDS molecules did not show a positive correlation with their electrophoretic mobilities. Moreover, it was found that the segment at positions 22-38 had the greatest capacity to bind with SDS than other segments of cobrotoxin, but guanidination of Lys residues of this peptide fragment did not significantly change its migration. These results suggest that the SDS-binding capacity of cobrotoxin is not solely responsible for its anomalous mobility in SDS gel, and that the effects may arise from the electrostatic interactions between Lys residue and carboxylated groups.

Cytotoxin-III from Naja naja atra (CTX) was chemically modified at either or both of its two methionine residues: Over 50% oxidation of methionine-26 occurred with a 1:1 molar ratio of chloramine-T:methionine; at a 5:1 molar ratio, methionine-26 was almost completely oxidized, while methionine-24 was modified only 26%; at a 10:1 molar ratio, both methionines were completely oxidized. Each oxidized derivative demonstrated a lower toxicity toward T-cells than toward heart cells. Conversely, binding to heart cells was affected more than binding to T-cells. Cyanogen bromide cleaved native CTX at both methionines, excising phenyl-alanine-25 and methionine-26 and converting methionine-24 to homoserine lactone. This treatment of CTX eliminated cytotoxicity toward both heart and T-cells, but had only a modest effect upon T-cell binding, as had 50% oxidation of methionine-26, suggesting that CTX lytic and binding regions may be distinct. A selective loss in heart cell binding following oxidation of methionine-24 further suggests that different parts of CTX may interact with the two types of target cells. Perturbation of the relatively flat hydrophobic surface of the CTX' triple-stranded beta-sheet could result from the introduction of negative charge due to methionine-24 oxidation. Alternatively, amino acid side chain participation in a CTX binding domain may be altered by the potential formation of a new hydrogen bond between tyrosine-51 and methionine-24 sulfoxide, as revealed by computer modeling of the completely oxidized CTX derivative.

In order to assess the manner in which the structural state of a protein affects the results of an ELISA, the antigenic reactivities of cobrotoxin and reduced and S-carboxymethylated (RCM-) cobrotoxin with anti-RCM-cobrotoxin antibodies were studied. The results of competitive enzyme-linked immunoassay showed that the affinity of RCM-cobrotoxin for anti-RCM-cobrotoxin antibodies was higher than that of cobrotoxin. However, the cobrotoxin-coated wells had a greater reactivity towards anti-RCM-cobrotoxin antibodies than against RCM-cobrotoxin-coated wells. The lower reactivity observed with RCM-cobrotoxin-coated plates could be improved by adding 0.01% glutaraldehyde during the coating procedure. Studies on the antigenic structures of RCM-cobrotoxin showed that it contained an immunodominant epitope at residues 22-38. Moreover, the N-terminal and C-terminal regions of RCM-cobrotoxin encompassed other antigenic determinants which exhibited low reactivities towards anti-RCM-cobrotoxin antibodies. After removal of the antibodies against residues 22-38 of cobrotoxin from anti-RCM-cobrotoxin antibodies by passage through an affinity column, the remaining antibodies exhibited a similar reactivity towards cobrotoxin and RCM-cobrotoxin. The antibodies against residues 22-38 retained a little reactivity with the RCM-cobrotoxin-coated wells. These results suggest that the structural determinants of cobrotoxin and RCM-cobrotoxin for binding to the microtitre plates differ. Unlike RCM-cobrotoxin, the loop II structure of cobrotoxin encompassing residues 22-38 is not exclusively involved in the binding of cobrotoxin to microtitre plates.

An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar beta-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected alpha/beta fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin alpha from the acetylcholine receptor at 10(-5) M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.

The proton resonances of cytotoxin II from Naja naja oxiana were sequentially assigned in 2D 1H NMR spectra for all of its 60 amino acid residues of both major and minor components of the spectra. The presence of the minor component was shown to be due to a conformational heterogeneity of the cytotoxin. The proton-deuterium exchange rates of amide groups were measured in 2H2O at a pH of 5.0 and at 10 degrees C. Experimental data obtained (d-connectivities, H-NC alpha-H coupling constants and long-range NOEs) allowed for the determination of the secondary structure of the two cytotoxin conformers. Both conformer structures contain two antiparallel beta-sheets. The first beta-sheet involves two antiparallel beta-strands comprising the residues 2-5 and 10-13. The second sheet involves three antiparallel strands consisting of the residues 20-26, 35-39, and 49-55; its peripheral beta-strands are connected by cross-over. The most striking structural difference between these conformers is the nature of the beta-turn 6-9, which has trans- and cis-forms of the Val7-Pro8 peptide bond in the major and minor conformer, respectively. The structures of other beta-turns, some of which are ascribed to standard types, and the C-termini are almost the same for the both conformers.

The presence of multiple nicotinic acetylcholine receptor (AchR)-binding proteins and phospholipases A2 was detected in the venom of a member of the Elapinae subfamily, Micrurus nigrocinctus nigrocinctus. Multi-step chromatographies were used to isolate four AchR-binding proteins (Mnn-9, Mnn-4, Mnn-3C and Mnn-1A) and five basic PLA2s (nigroxins A, B, C1, C2 and C3). The Micrurus AchR-binding proteins are antigenically and structurally related to short- and long-chain alpha-neurotoxins from Naja. The nigroxins are antigenically similar and constitute a new antigenic subclass of PLA(2)s. Nigroxins A and B are class I PLA(2)s, structurally more related to enzymes from Bungarinae than to those from Hydrophinae/Laticaudinae. These data contribute to clarify the relationships between Micrurus venom proteins and other elapid toxins and may be useful to improve the neutralizing efficiency of antivenoms.

The cationic groups of arginine and lysine residues in alpha-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) and p-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.