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NMR Solution Structures of δ-Conotoxin EVIA from Conus ermineus That Selectively Acts on Vertebrate Neuronal Na+ Channels

2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês

10.1074/jbc.m309594200

ISSN

1083-351X

Autores

Laurent Volpon, Hung Lamthanh, Julien Barbier, Nicolas Gilles, Jordi Molgó, Andre Ménèz, Jean‐Marc Lancelin,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

δ-Conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. δ-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6), without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu12-Pro13 cis/trans isomerism, a Pro13 → Ala δ-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A δ-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice. δ-Conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. δ-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6), without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu12-Pro13 cis/trans isomerism, a Pro13 → Ala δ-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A δ-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice. The new δ-conotoxin EVIA (δ-EVIA), 1The abbreviations used are: δ-EVIA, δ-conotoxin EVIA from Conus ermineus; DQF-COSY, double quantum-filtered correlation spectroscopy; Hyp, hydroxyproline; r.m.s.d., root mean square atomic deviation; HPLC, high pressure liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; S-cam, S-carboxamidomethyl; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy. a 32-amino acid conopeptide isolated from the venom of Conus ermineus, is the first conotoxin demonstrated to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6) without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel subtypes (1Barbier J. Lamthanh H. Le Gall F. Favreau P. Benoit E. Chen H. Gilles N. Ilan N. Heinemann S.F. Gordon D. Ménez A. Molgó J. J. Biol. Chem. 2004; 279: 4680-4685Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This important recent discovery makes δ-EVIA a unique tool to study the modulation mechanisms of neuronal Na+ channels. As a consequence, δ-EVIA may also serve as a new lead molecule for the design of new drugs to treat neurological diseases characterized by defective nerve conduction, especially those causing an axonal demyelinization (2Kaji R. Muscle Nerve. 2003; 27: 285-296Crossref PubMed Scopus (130) Google Scholar, 3Hattori N. Yamamoto M. Yoshihara T. Koike H. Nakagawa M. Yoshikawa H. Ohnishi A. Hayasaka K. Onodera O. Baba M. Yasuda H. Saito T. Nakashima K. Kira J. Kaji R. Oka N. Sobue G. Brain. 2003; 126: 134-151Crossref PubMed Scopus (186) Google Scholar). Nerve conduction could be facilitated by specific inhibition of Na+ channel inactivation. The knowledge of the detailed three-dimensional structure is therefore the first step necessary to understand the structure-activity relationships of this new lead conotoxin. Despite a low sequence identity with the κ-, ω-, and δ-conotoxins, δ-EVIA clearly belongs to the four-loop family of conotoxins characterized by a similar cysteine pairing giving a conserved 3-disulfide framework as shown in Fig. 1. Until now, the three-dimensional structure of 10 conotoxins belonging to this family was already determined as follows: the conotoxin κ-PVIIA (20Savarin P. Guenneugues M. Gilquin B. Lamthanh H. Gasparini S. Zinn-Justin S. Menez A. Biochemistry. 1998; 37: 5407-5416Crossref PubMed Scopus (80) Google Scholar) targeting potassium channels; conotoxins ω-MVIIA (21Kohno T. Kim J.I. Kobayashi K. Kodera Y. Maeda T. Sato K. Biochemistry. 1995; 34: 10256-10265Crossref PubMed Scopus (107) Google Scholar), ω-MVIIC (22Farr-Jones S. Miljanich G.P. Nadasdi L. Ramachandran J. Basus V.J. J. Mol. 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Structure. 1997; 5: 571-583Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) targeting skeletal muscle sodium channels; conotoxin TVIIA (28Hill J.M. Alewood P.F. Craik D.J. Eur. J. Biochem. 2000; 267: 4649-4657Crossref PubMed Scopus (13) Google Scholar); and the most recent conotoxin δ-Tx-VIA (12Kohno T. Sasaki T. Kobayashi K. Fainzilber M. Sato K. J. Biol. Chem. 2002; 277: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The latter δ-TxVIA belongs to a specific class of conotoxins that affect Na+ channel inactivation exclusively in mollusks but exhibits high affinity to rat brain synaptosomes (15Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 16Yanagawa Y. Abe T. Satake M. Odani S. Suzuki J. Ishikawa K. 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Biochemistry. 1995; 34: 4913-4918Crossref PubMed Scopus (96) Google Scholar), their three-dimensional solution structures reveal a common scaffold consisting of a small β-hairpin structure and several types of tight turns. All conotoxins of this group exhibit a rather well defined backbone conformation, stabilized by a number of hydrogen bonds located in the different secondary structures, and by the three disulfide bridges. The scaffold forms the classical "cystine-knot" motif known for toxic and inhibitory polypeptides (29Pallaghy P.K. Nielsen K.J. Craik D.J. Norton R.S. Protein Sci. 1994; 3: 1833-1839Crossref PubMed Scopus (470) Google Scholar). A remarkable feature of δ-EVIA is the length of the loop 2 is made of nine residues instead of six for the other conotoxins. A variability in the cystine-knot scaffold, both in sequence and length, was already described for the loop 4 of conotoxin μ-GS (27Hill J.M. Alewood P.F. Craik D.J. Structure. 1997; 5: 571-583Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) or the δ-atracotoxin-Hv1 (30Fletcher J.I. Chapman B.E. Mackay J.P. Howden M.E. King G.F. Structure. 1997; 5: 1525-1535Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Fig. 1) but not for the loop 2. We describe here the three-dimensional NMR structure in aqueous solution of δ-EVIA. The specific difference with the solution conformation of the other members of the four-loop family of conotoxins is located in loop 2, which is characterized by an apparent low conformational order corroborated by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR chemical shift time scale. Both cis Leu12-Pro13 and trans Leu12-Pro13 bonds were separately solved based on two separate sets of experimental restraints. For comparison purposes, a Pro13 → Ala single mutant was also prepared. The NMR data for P13A mutation are consistent with a 12-13 peptide bond purely trans, whereas the global structure of the toxin is maintained. The binding of the P13A δ-EVIA to rat brain synaptosomes as well as its activity, measured by intracerebroventricular injection to mice, is reduced about 2-fold. Toxin synthesis-solid phase synthesis of the linear δ-EVIA and P13A δ-EVIA peptides were done with an ABI 430A synthesizer (PerkinElmer Life Sciences), using Fmoc chemistry with 1,3-dicyclohexylcarbodiimide/HOAt as the condensation reagent (31Carpino L.A. El-Faham A. Minor C. Albericio F. J. Chem. Soc. Chem. Commun. 1994; : 201-203Crossref Google Scholar) and 0.1 mmol of Rink amide resin. After the last Fmoc-amino acid coupling, the Fmoc group was kept bound to the N-terminal amino acid of the linear peptide in order to simplify the read out of the HPLC profile during its purification. The N-α-Fmoc linear peptide was cleaved from the resin and purified with a reverse phase preparative column (Vydac, 218TP, 250 × 25 mm). The peptide was eluted by a linear gradient of A and B solvents, 30-100% B in 70 min; A is an aqueous solution of 0.1% (v/v) trifluoroacetic acid, and B is an aqueous solution containing 60% (v/v) acetonitrile and 0.1% trifluoroacetic acid. After elution, the major component (151 mg), corresponding to the target Fmoc peptide (mass spectrum, m/z 3515.32), was deprotected by incubation (5 min, 22 °C) with a mixture composed of N,N-dimethyl formamide (5 ml) containing 20% piperidine (v/v) and 1% 1,8-diazabicyclo[5.4.0]undec-7-ene (v/v) and 5 ml of mercaptoethanol (72 mmol). After deprotection, the medium was diluted with deionized water (final volume ∼500 ml) and acidified to pH 4 with trifluoroacetic acid, and the linear peptide was desalted by reverse phase preparative HPLC, as described previously. A pool of the δ-EVIA-containing fractions (∼100 ml) was diluted with deionized water (final volume, ∼500 ml) containing the redox couple cysteine/cystine, 2/0.5 mmol. The volume was adjusted to 1 liter with degassed buffer (0.1 m NH4SO4, 0.1 m ammonium acetate, 1 mm EDTA, pH 8.5), and the solution was adjusted to pH 8.5 with NH4OH and renatured by incubation (48 h at 4 °C, followed by 12 h at 22 °C). The mixture of oxidized peptides was adjusted to pH 4 with trifluoroacetic acid, loaded on reverse phase preparative column (Vydac, 218TP, 250 × 25 mm), and eluted with the acetonitrile gradient described previously. Based on HPLC co-elution experiments and electrospray ionization mass spectrometry, δ-EVIA was identified as a minor product under the oxidation conditions used. The peptide was further purified with a semi-preparative column (Zorbax SB C18, 250 × 9.4 mm). Disulfide Pairing Assignment—δ-EVIA disulfide pairing pattern was determined after partial reduction with tris(2-chloroethyl)phosphate (40 °C, 5 min), as reported previously (32Gray W.R. Protein Sci. 1993; 2: 1732-1748Crossref PubMed Scopus (236) Google Scholar). The reverse phase chromatographic profile of the mixture is shown in Fig. 2A. The fingerprint pattern displayed a huge peak of nonreduced δ-EVIA and three intermediates with one or two open disulfide bonds (peaks A-C). The fully reduced peptide (SH)6 eluted earlier than the nonreduced δ-EVIA and the three intermediates. After the intermediates in peaks A-C were alkylated with a large excess of iodoacetamide and purified by reverse phase HPLC, the amino acid sequences of their Cys (S-carboxamidomethyl) derivatives were determined (Fig. 2B). Sequencing the Cys (S-cam) derivative in peak A indicated SS bridging of Cys3 and Cys21. Sequencing the Cys (S-cam) derivative in peak B revealed an increased signal for Cys (S-cam) at cycles 3, 10, 21, and 25. Thus, peak B has the disulfide bridge Cys10, Cys25 in addition to the disulfide bridge Cys3, Cys21. Analysis of peak C yielded a significant increase in the Cys (S-cam) signal at cycles 10 and 25, due to the open disulfide bridge Cys10, Cys25. Therefore, we deduce SS bridging between Cys20, Cys29,in addition to the detected Cys3, Cys21 and Cys10, Cys25 disulfide bonds. The cystine framework of δ-EVIA was summarized as Cys3, Cys21, Cys10, Cys25, and Cys20, Cys29. Radioiodination and Binding Assays—δ-Conotoxin TxVIA was radio-iodinated by using 1 nmol of toxin, 0.5 mCi of carrier-free Na125I in a potassium phosphate buffer, pH 7.25, containing H2O2 (10 μl of 1:50,000 solution) and lactoperoxidase (0.7 unit, EC 1.11.1.7 from bovine milk) for a 2-min incubation time. The monoiodotoxin was purified on a Vydac C18 column. Rat brain synaptosomes were prepared from adult Sprague-Dawley rats (300 g), according to the method described by Kanner (33Kanner B.J. Biochemistry. 1978; 17: 1207-1211Crossref PubMed Scopus (212) Google Scholar). Equilibrium competition assays were performed using increasing concentrations of unlabeled toxins in the presence of a constant low concentration of the radioactive toxin. Competition binding experiments were analyzed by the program Kaleidagraph (Synergy Software) by using a non-linear Hill equation (for IC50 determination). The Ki values of δ-EVIA were calculated by the equation Ki = IC50/(1 + (L*/Kd)) where L* is the concentration of the hot δ-TxVIA, and Kd is its dissociation constant (34Cheng Y.C. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12330) Google Scholar). Standard binding medium composition was (in mm) as follows: choline Cl 130, CaCl2 1.8, KCl 5, MgSO4 0.8, HEPES 50, glucose 10, and 2 mg/ml bovine serum albumin. Following incubation for the designated times, the reaction was terminated by dilution with 2 ml of ice-cold wash buffer of the following composition (in mm): choline Cl 140, CaCl2 1.8, KCl 5.4, MgSO4 0.8, HEPES 50, pH 7.2, 5 mg/ml bovine serum albumin. Separation of free from bound toxin was achieved by rapid filtration under vacuum using Whatman GF/C filters preincubated with 0.3% polyethyleneimine. The filter discs were then rapidly washed twice with 2 ml of buffer. Nonspecific toxin binding was determined in the presence of a high concentration of the unlabeled toxin. Biological Activity—To quantify the biological activity of native and synthetic conotoxins, Swiss-Webster mice (≈15 g) were injected intracerebroventricularly with a stereotaxic system (Harvard/ASI Apparatus, UK). The ED50 value was defined as the dose that produces hyper-activity in 50% of the tested animals within 12 h postinjection. NMR Spectroscopy—δ-EVIA was dissolved at 2.2 mm (4 mg/550 μl) in either 90% H2O, 10% D2O, or 100% D2O. pH was adjusted to 3.0 (direct uncorrected pH-meter reading) using microliter increments of 0.1 n HCl. The P13A analog was prepared at 0.6 mm in 90% H2O, 10% D2O, pH 3.0. NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer using a 5-mm (1H, 13C, 15N) triple-resonance probe head, equipped with a supplementary self-shielded z gradient coil. Spectra were processed using Bruker XWINNMR and GIFA V.4 (35Pons J.L. Malliavin T.E. Delsuc M.A. J. Biomol. NMR. 1996; 8: 445-452Crossref PubMed Scopus (228) Google Scholar) software. The solvent signal was suppressed with the WATERGATE sequence using a 3-9-19 pulse sequence with z gradient (36Piotto M. Saudek V. Sklenár V. J. Biomol. NMR. 1992; 2: 661-666Crossref PubMed Scopus (3539) Google Scholar, 37Sklenár V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Sect. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar). The DQF-COSY experiment (38Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar) was recorded at 283 K in H2O with a very low power presaturation of the water resonance during the recycle delay to minimize the radiation damping effect. TOCSY/HOHAHA experiments (39Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3112) Google Scholar, 40Davies D.G. Bax A. J. Am. Chem. Soc. 1985; 107: 2820-2821Crossref Scopus (1073) Google Scholar) were collected in both H2O and D2O at 283 K with a spin-lock time of 40 and 80 ms and at 290 and 297 K with a spin-lock time of 80 ms. An MLEV pulse sequence was used for the isotropic mixing arranged as the clean-TOCSY scheme (41Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1199) Google Scholar). NOESY spectra (42Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar, 43Macura S. Hyang Y. Suter D. Ernst R.R. J. Magn. Reson. 1981; 43: 259-281Google Scholar) were collected at 283, 290, and 297 K with a 1.5-s recycle time and a 150-ms mixing time. Supplementary NOESY spectra were collected at 283 K in H2O and D2O with mixing time of 75 and 250 ms, respectively. In these NOESY experiments, a selective flip-back pulse was applied on water resonance, and gradients were added during t1 to minimize the radiation damping effect (44Lippens G. Najib J. Wodak S.J. Tartar A. Biochemistry. 1995; 34: 13-21Crossref PubMed Scopus (116) Google Scholar). Except for the NOESY experiment at 283 K in H2O where 96 scans per t1 increments were accumulated, spectra were registered with 512 (t1) × 1024 (t2) complex data points and 32 scans per t1. The quadrature detection in the t1 dimension was achieved using the States-TPPI method (45Marion D. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1989; 85: 393-399Google Scholar). Chemical shifts were quoted relative to the solvent (H2O) chemical shift at the respective temperature (4.92 ppm at 283 K). The spectral width of all experiments was 10.96 ppm (5482.5 Hz) with a carrier frequency on-resonance with the water resonance. For the P13A conotoxin δ-EVIA, the TOCSY spectrum was collected with a spin-lock time of 80 ms, and NOESY spectrum was recorded with a 150-ms mixing time. Deuterium exchange of labile protons was monitored just after dissolution of the lyophilized sample from H2O at pH 3.0 in neat D2O. The residual NH signal was followed with time at 283 K by analysis of TOCSY spectra recorded at 0.5, 1, 3, 19, 36, and 54 h. Experimental NMR Restraints—Interproton-distance constraints were classified into four categories according to the cross-peak intensity of the NOESY spectrum at 283 K and 150-ms mixing time in H2O and D2O. Upper bounds were fixed at 2.7, 3.3, 5.0, and 6.0 Å for strong, medium, weak, and very weak correlations, respectively. The lower bound for all restraints was fixed at 1.8 Å, which corresponds to the sum of the hydrogen van der Waals' radii. The calibration for the NOE intensities was achieved using the cross-peak intensity Hδ-Hϵ of Tyr7. Pseudo-atom corrections (46Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley Interscience, New York1986Crossref Google Scholar) of the upper bounds were applied for magnetically equivalent aromatic protons (+2 Å) and unresolved diastereotopic methyl or methylene protons (+ 1 Å). 0.3 Å was added to NOEs involving amide protons. Dihedral angle restraint χ1 was deduced from the stereospecific assignments of diastereotopic β-protons (±40° from the ideal staggered conformation) (47Hyberts S.G. Marki W. Wagner G. Eur. J. Biochem. 1987; 164: 625-635Crossref PubMed Scopus (202) Google Scholar, 48Wagner G. Braun W. Havel T.F. Schaumann T. Go N. Wüthrich K. 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J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar) and promotif (52Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (999) Google Scholar) and displayed using MolMol version 2.4 (53Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar) and Molscript (54Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). The structure was generated by the hybrid distance geometry dynamically simulated annealing method (55Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (772) Google Scholar, 56Kuszewski J. Nilges M. Brünger A.T. J. Biomol. NMR. 1992; 2: 33-56Crossref PubMed Scopus (210) Google Scholar). In a first stage, the substructures generated using metric matrix distance geometry algorithms were regularized and refined by a high temperature simulated annealing protocol, using the parallhdg.pro force field of X-PLOR. The non-bonded van der Waals' interactions were represented by a simple repulsive quadratic term (55Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (772) Google Scholar, 57Brünger A.T. Karplus M.J. Acc. Chem. Res. 1991; 24: 54-61Crossref Scopus (114) Google Scholar). The experimental distance restraints were represented as a soft asymptotic potential, and electrostatic interactions were ignored. The force constant associated with the distance restraints was kept to 50 kcal·mol-1·Å-2 throughout the protocol. One cycle of simulated annealing refinement consisted of 1,500 steps of 3 fs at 1,000 K followed by 3,000 cooling steps of 1 fs from 1,000 to 300 K. At the end, each structure was subjected to 1,500 steps of conjugate gradient energy minimization. In the second stage of the calculation, structures with good experimental and geometric energies were further refined using the full CHARMM22 force field of X-PLOR. In this stage of the calculation, the non-bonded interactions such as electrostatic interactions and van der Waals' interactions (described by the Lennard-Jones empirical energy function) were taken into account. An approximate solvent electrostatic screening effect was introduced by using a distance-dependent dielectric constant and by reducing the electric charges of the formally charged amino acid side chain (Asp and Lys) to 20% of its nominal charges defined in the CHARMM22 force field. The force constant used for the NOE potential was reduced to 25 kcal·mol-1·Å-2. After 1,500 steps of conjugate gradient energy minimization, the dynamic was initiated at 750 K. The system was equilibrated for 0.5 ps with an integration step of 1 fs and then coupled to a heat bath at 750 K, and the molecule was allowed to evolve for 10 ps before a slow cooling to 300 K for a period of 5.4 ps and allowed to evolve again for 15 ps. Finally, the structures were energy-minimized by 1,500 steps of the conjugate gradient algorithm. δ-EVIA was synthesized using Fmoc chemistry, based on its amino acid sequence. The Nα-Fmoc linear/denatured and reduced peptide was obtained in high yield (151/350 mg of calculated Fmoc peptide). Its folding/renaturation was the limiting step in the synthesis of bioactive δ-EVIA. Typically, the folding procedure yielded only 5-10 mg of synthetic and bioactive δ-EVIA (3.3-6.6% yield, based on 151 mg of Fmoc linear peptide). The HPLC pattern of the preparation obtained after the folding procedure indicated the presence of a large quantity of monomer isomass peptides (data not shown), probably because of misfolding of the linear, reduced peptide. Synthetic δ-EVIA co-eluted with native δ-EVIA (Fig. 2C), and the predicted MW of synthetic δ-EVIA was confirmed by ESI-MS (observed MH+ 3288.1; calculated MH+ 3288.4). Sequence Resonance Assignments of the Spin Systems—From the DQF-COSY and TOCSY spectra, we first noticed the presence of 53 amide protons (HN), instead of the 30 expected. The sequence-specific resonance assignment (46Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley Interscience, New York1986Crossref Google Scholar) was undertaken and revealed two distinct NOESY walking with only Asp1, Asp2, Lys5, Hyp6, Gly8, Cys21, and Leu32 common to the two systems. The dαN connectivities proceeded unambiguously for the two sets of resonances except for Cys21/Ser22. Tyr7, Phe9, Pro13, Ala24, and Ala30 spin systems were used as starting points for the sequential assignment due to their specific pattern. In one of the two systems, the Leu12-Pro13 peptide bond was unambiguously assigned to the cis peptide conformation due to the observation of a strong dαα and a weak dαδ in NOESY spectra. For the second system, the peptide bond conformation was unambiguously assigned to the trans conformation due to the observation of a strong dαδ with no dαα. Then the two systems clearly resulted from a Leu12-Pro13 cis/trans mixture of δ-EVIA in slow exchange on the time scale of the NMR chemical shift. No exchange peaks between the two conformers were detected either in the TOCSY or the NOESY experiments (Fig. 3). The chemical shift differences (where Δδc-t indicates difference of the chemical shifts between cis and trans conformers) observed in the NM

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