Biomedical subjects

C Vita

Publications and source records attributed to C Vita.

At least 37 records · Page 2Linked to original sources

PubMed1997

On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.

Amino Acid Sequence↗

PubMed1997

Engineering novel proteins by transfer of active sites to natural scaffolds.

Novel functional proteins have been generated by the transfer of active sites to structurally homologous proteins and to new structural contexts. The most successful examples of these approaches succeeded in providing effective new tools in biochemistry and protein chemistry and in suggesting new models in drug design.

Animals↗

PubMed1996

Transfer of a beta-hairpin from the functional site of snake curaremimetic toxins to the alpha/beta scaffold of scorpion toxins: three-dimensional solution structure of the chimeric protein.

The alpha/beta scorpion fold is shared by scorpion toxins, insect defensins, and plant thionins. This small and functionally versatile template contains an alpha-helix and a triple beta-sheet linked by three disulfide bridges. With the view to introduce novel functional centers within this fold, we replaced the sequence (the cysteines and glycines excepted) of the original beta-hairpin of a scorpion toxin by the sequence of a beta-hairpin that forms part of the site by which snake neurotoxins bind to nicotinic acetylcholine receptors (AcChOR). The resulting chimeric protein, synthesized by chemical means, binds to AcChOR, though with a lower affinity than the snake toxins [Drakopoulou; E., Zinn-Justin, S., Guenneugues, M., Gilquin, B., Ménez, A., & Vita, C. (1996) J. Biol. Chem. 271, 11979-11987]. The work described in this paper is an attempt to clarify the structural consequences associated with the transfer of the beta-hairpin. We report the determination of the three-dimensional solution structure of the chimeric protein by proton NMR spectroscopy and molecular dynamics calculations. Comparison of the structure of the chimera with those of the scorpion alpha/beta toxin and of the snake neurotoxin shows that (i) the new protein folds as an alpha/beta motif and (ii) the beta-hairpins of the chimera and of the curaremimetic toxin adopt a similar conformation. A closer inspection of the differences between the structures of the original and transferred beta-hairpins allows rationalization of the biological properties of the chimera.

Amino Acid Sequence↗

PubMed1996

Changing the structural context of a functional beta-hairpin. Synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion alpha/beta scaffold.

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.

Amino Acid Sequence↗

PubMed1996

Determination of the C-terminal form of an anemone toxin using capillary electrophoresis and mass spectrometry.

To identify the form of the C-terminal amino acid of a sea anemone toxin, the native protein was compared with two synthetic proteins comprising the same sequence and a free or an amide C-terminal form. Using electrospray ionization-mass spectrometry, capillary electrophoresis and the coupling of both techniques, we assigned the C-terminus of the native protein to be in the free carboxyl form.

Animals↗

PubMed1995

A particularly labile Asp-Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage.

The single Asp53-Pro54 bond of the MTX2 toxin from the mamba snake Dendroaspis angusticeps is rapidly and efficiently cleaved in acidic solution (pH 1.5-2.5) at 45 degrees C. Unfolding of the toxin slows down the cleavage reaction by several times. Modelling studies indicate that the native toxin conformation can catalyse the Asp53-Pro54 bond cleavage. The implications of this study are: (i) cleavage of Asp-Pro bond for sequence determination may occur better in absence than in presence of denaturant, (ii) mild acid conditions, commonly used in NMR structure determinations, may irreversibly affect the structural integrity of Asp-Pro containing peptides and proteins.

Amino Acid Sequence↗

PubMed1995

Chemical synthesis, structural and functional characterisation of noxiustoxin, a powerful blocker of lymphocyte voltage-dependent K+ channels.

Two forms of the Centrudoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K+ channels with 2 nM affinity, but does not affect the Ca(2+)-activated K+ channels. This toxin, because of its high affinity and specificity for voltage-gated K+ channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.

Amino Acid Sequence↗

PubMed1995

Scorpion toxins as natural scaffolds for protein engineering.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.

Amino Acid Sequence↗

PubMed1995

Structure-activity studies on scorpion toxins that block potassium channels.

Scorpion venoms contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca(2+)-activated K+ channels and for dendrotoxin-sensitive voltage-dependent K+ channels. The structural features that determine the specificity of binding to different channel types are not known. We investigated this using natural and synthetic scorpion toxins. We have tested the effects of charybdotoxin (CTX) and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin (IbTX), and kaliotoxin (KTX) from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively, and synthetic variants of CTX, namely CTX2-37, CTX3-37, CTX4-37, and CTX7-37, on a Ca(2+)-activated K+ current (IK-Ca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, 125I-DpI, to voltage-dependent K+ channels on rat brain synaptosomal membranes. The native toxins contain 37-38 amino acid residues, they are over 30% identical in sequence (CTX and IbTX are 68% identical), and they have similar three-dimensional conformations. All toxins, except IbTX, displaced 125I-DpI from its synaptosomal binding sites: Lqh 18-2 (Ki = 0.25 nM), KTX (Ki = 2.1 nM), CTX (Ki = 3.8 nM), CTX2-37, (Ki = 30 nM), Lqg 15-1 (Ki = 50 nM), CTX3-37 (Ki = 60 nM), CTX4-37 (Ki = 50 nM), CTX7-37 (Ki = 105 nM). IbTX had no effect at 3 microM. When variants of CTX with deletions at the N-terminal portion were tested for their activity on IK-Ca on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX3-37 and CTX4-37 were ineffective at 100 nM; and CTX7-37 was ineffective at up to 1 microM. IbTX and CTX (100 nM) completely blocked IK-Ca, but KTX (100 nM) did not affect the nerve terminal IK-Ca. Different residues appear to be important for interactions of the toxins with different K+ channels. IbTX did not displace dendrotoxin binding, but it did block IK-Ca, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the IK-Ca of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of IK-Ca at the motor nerve terminal: it is truncated in the inactive synthetic CTX variants; and it is positively charged at lysine-6 in KTX (which is inactive), but negatively charged in IbTX and neutral in CTX.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Synthesis of a metal binding protein designed on the alpha/beta scaffold of charybdotoxin.

The alpha/beta scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2% yield. Circular dichroism analysis showed that the newly designed protein is folded in a alpha/beta structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu2+ and Zn2+ ions bind with high affinity to the newly engineered protein. These results demonstrate that the alpha/beta fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins.

Amino Acid Sequence↗

PubMed1993

Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation.

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Amino Acid Sequence↗

PubMed1993

Toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture.

The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K+ against Na+ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l NaCl in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (Po) in a concentration-dependent fashion (IC50 = 0.8 mg/l; n = 3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC50 = 7.3 x 10(-9) M (n = 5); iberiotoxin (IbTX) IC50 = 5.5 x 10(-7) mol/l (n = 3); and kaliotoxin (KTX) IC50 = 4.8 x 10(-7) mol/l (n = 3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC50 = 1.2 x 10(-8) mol/l, n = 4). Neither Dendroaspis polylepis venom nor purified alpha dendrotoxin modified Po even at high concentrations (20 mg/l and 10(-6) mol/l respectively). Apamin, which blocked the small-conductance K+ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.

A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.

Amino Acid Sequence↗

PubMed1992

Total synthesis of horse heart cytochrome C.

A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis of an entire protein.

Amino Acid Sequence↗

PubMed1990

Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments.

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.

Amino Acid Sequence↗

PubMed1990

Lack of correlation between structural properties and biological activity of two cross-reacting cytochrome c T cell epitopes as determined by circular dichroism measurements.

Horse and tuna cytochrome c synthetic peptides 39-53 can equally well stimulate a cytochrome c specific T cell hybridoma. The amino acid sequence of the two peptides differs mainly for the presence of a helix-breaking proline and a helix-forming glutamic acid in position 44. Circular dichroism studies of these two peptides revealed that their conformation could be drastically depending on the solvent used. These data place some limits on the correlation between structural data and biological activity.

Animals↗

PubMed1990

Solid-phase synthesis of the native sequence of horse heart cytochrome c-(66-104)-nonatriacontapeptide and of a C-terminal carboxyamide analog selectively modified at Met-80.

The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.

Amino Acid Sequence↗

PubMed1990

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.

Alcohols↗