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A crucial mechanism to the formation of native, fully functional, 3D structures from local secondary structures is unraveled in this study. Through the introduction of various amino acid substitutions at four canonical β-turns in a three-fingered protein, Toxin α from Naja nigricollis, we found that the release of internal entropy to the external environment through the globally synchronized movements of local substructures plays a crucial role. Throughout the folding process, the folding species were saturated with internal entropy so that intermediates accumulated at the equilibrium state. Their relief from the equilibrium state was accomplished by the formation of a critical disulfide bridge, which could guide the synchronized movement of one of the peripheral secondary structure. This secondary structure collided with a core central structure, which flanked another peripheral secondary structure. This collision displaced the internal thermal fluctuations from the first peripheral structure to the second peripheral structure, where the displaced thermal fluctuations were ultimately released as entropy. Two protein folding processes that acted in succession were identified as the means to establish the flow of thermal fluctuations. The first process was the time-consuming assembly process, where stochastic combinations of colliding, native-like, secondary structures provided candidate structures for the folded protein. The second process was the activation process to establish the global mutual relationships of the native protein in the selected candidate. This activation process was initiated and propagated by a positive feedback process between efficient entropy release and well-packed local structures, which moved in synchronization. The molecular mechanism suggested by this experiment was assessed with a well-defined 3D structure of erabutoxin b because one of the turns that played a critical role in folding was shared with erabutoxin b., Competing Interests: The authors have declared that no competing interests exist.

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.

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.

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.

In a previous paper, the systematic epitope screening of a snake curaremimetic toxin or toxin a was described by this group using a panel of synthetic octadecapeptides. The disulphide cyclized peptide (Cys-23,40)(23-40) corresponding to loop II of the native toxin was found to elicit, with no linkage to a carrier, neutralizing antisera against the toxin. We have now undertaken the conformational study of this immunogenic disulphide cyclized peptide by CD and FTIR. The CD study of the peptide was carried in aqueous solution under various conditions (pH, temperature, presence of micelles) and in trifluoroethanol solution. Low temperature, SDS micelles and trifluoroethanol were found to induce a beta-sheet formation (16 to 39%). FTIR spectra of the peptide in the solid state (dry film) and in D2O solution or deuterated-TFE solution (hydrated film) displayed some characteristic bands indicating the presence of beta-sheet (1623 cm-1) and beta-turn (1637 cm-1; 1694 cm-1). These studies indicate that the immunogenic disulphide cyclized peptide (23-40) can adopt in solution an ordered structure.

We have investigated the luminescence and optically detected magnetic resonance (ODMR) of the highly homologous snake venom neurotoxins alpha-bungarotoxin (BgTX), alpha-cobratoxin (CbTX), and cobrotoxin (CoTX) in frozen aqueous glasses. The phosphorescence intensity and lifetime of the single invariant tryptophan, Trp29, are found to be diminished in BgTX and CbTx relative to CoTX both at 77 K and at 4.2 K. Selective reduction of the Cys30-Cys34 disulfide proximal to Trp29 in BgTX and CbTX, that is absent in CoTX, results in the enhancement of the phosphorescence to fluorescence intensity ratio of Trp29 and identifies this disulfide as the source of the triplet-state quenching. Variations of the phosphorescence parameters are observed for differently frozen BgTX and CbTX samples. We argue that this observation is consistent with conformational flexibility in the region of Trp29. For BgTX and CbTX, changing the wavelength of excitation from 285 to 300 nm results in a small bathochromic phosphorescence shift of 0.4 nm, an average decrease in the lifetime, and a change in the polarity of the normally positive D-E ODMR signal. From the small excitation-dependent emission shift, we infer that Trp29 is in a relatively hydrophobic environment. The excitation-dependent changes in lifetime and ODMR signal parameters arise from subtle heterogeneity in the disposition of Trp29 with respect to Cys30-Cys34. We discuss the mechanism of disulfide-induced quenching of the Trp29 triplet state in BgTX and CbTX and argue that it most probably is due to electron transfer.

Analysis of C-alpha atom positions from cysteines involved in disulphide bridges in protein crystals shows that their geometric characteristics are unique with respect to other Cys-Cys, non-bridging pairs. They may be used for predicting disulphide connections in incompletely determined protein structures, such as low resolution crystallography or theoretical folding experiments. The basic unit for analysis and prediction is the 3 x 3 distance matrix for Cx positions of residues (i - 1), Cys(i), (i +1) with (j - 1), Cys(j), (j + 1). In each of its columns, row and diagonal vector--outer distances are larger than the central distance. This analysis is compared with some analytical models.