1H NMR study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27 degrees C. Individual pKa values have been obtained for the alpha-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 degrees C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27 degrees C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

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

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

Sequential 1H-NMR assignments and secondary structure of the sea anemone polypeptide anthopleurin-A

The sequence-specific assignment of resonances in the 500-MHz 1H-NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin-A, is described. The assignment procedure involved analysis of two-dimensional phase-sensitive multiple-quantum-filtered, double-quantum, homonuclear Hartmann-Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues. Resonances arising from a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Gooley, P. R. and Norton, R. S. (1985) Eur. J. Biochem. 153, 529-539] and provide the basis for a more accurate definition of the conformation of anthopleurin-A in aqueous solution. The secondary structure includes a four-stranded antiparallel beta-sheet encompassing residues 2-4, 21-23, 34-36 and 45-49, and possibly a beta-bulge located at Ser-19 and Gly-20. A type II beta-turn is formed by residues 30-33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro-41 appears to be unique to anthopleurin-A.     

Sequence-specific 1H NMR assignments and secondary structure of porcine motilin

The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.     

Sequence-specific 1H NMR assignments and secondary structure of eglin c

Sequence-specific nuclear magnetic resonance assignments were obtained for eglin c, a polypeptide inhibitor of the granulocytic proteinases elastase and cathepsin G and some other proteinases. The protein consists of a single polypeptide chain of 70 residues. All proton resonances were assigned except for some labile protons of arginine side chains. The patterns of nuclear Overhauser enhancements and coupling constants and the observation of slow hydrogen exchange were used to characterize the secondary structure of the protein. The results indicate that the solution structure of the free inhibitor is very similar to the crystal structure reported for the same protein in the complex with subtilisin Carlsberg. However, a part of the binding loop seems to have a significantly different conformation in the free protein.     

Sequence-specific 1H NMR assignments and solution structure of bovine pancreatic polypeptide.

Sequence-specific 1H NMR assignments for the 36 residue bovine pancreatic polypeptide (bPP) have been completed. The secondary and tertiary structure of bPP in solution has been determined from experimental NMR data. It is shown that bPP has a very well-defined C-terminal alpha-helix involving residues 15-32. Although regular secondary structure cannot be clearly defined in the N-terminal region, residues 4-8 maintain a rather ordered conformation in solution. This is attributed primarily to the hydrophobic interactions between this region and the C-terminal helix. The two segments of the structure are joined by a turn which is poorly defined. The four end residues both at the N-terminus and the C-terminus are highly disordered in solution. The overall fold of the bPP molecule is very closely similar to that found in the crystal structure of avian pancreatic polypeptide (aPP). The RMS deviation for backbone atoms of residues 4-8 and 15-32 between the bPP mean structure and the aPP crystal structure is 0.65 A, although there is only 39% identity of the residues. Furthermore, the average conformations of some (mostly from the alpha-helix) side chains of bPP in solution are closely similar to those of aPP in the crystal structure. A large number of side chains of bPP, however, show significant conformational averaging in solution.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

Pharmacological effects of two cytolysins isolated from the sea anemone Stichodactyla helianthus

Sticholysins I and II (St I/II) are cytolysins purified from the sea anemone Stichodactyla helianthus. In this study, we show their pharmacological action on guinea-pig and snail models in native and pH-denatured conditions in order to correlate the pharmacological findings with the pore-forming activity of both isoforms. In guinea-pig erythrocytes (N = 3), St II possessed higher haemolytic activity in comparison with St I and this activity was lost at an alkaline pH. In molluscan central neurons (N = 30), they irreversibly decreased the amplitude of the cholinergic response; St I (EC ₅₀ 0.6 µmolL^?1) was more potent than St II (EC₅₀ >6.6 µmolL^?1) and they both increased the duration of the action potential; these effects were absent at an alkaline pH. In guinea-pig isolated atrium (N = 25), both increased the amplitude of the contraction force, but St II was more potent than St I (EC₅₀ 0.03 µmolL^?1 and 0.3 µmolL^?1, respectively) and this effect persisted at an alkaline pH. In summary, both cytolysins have neuroactive and cardioactive properties. The main mechanism in molluscan neurons seems to be associated with the cytolytic activity of these molecules, whereas in guinea-pig atrium, the existence of an additional pharmacological mechanism might be contributing to the observed effect.     

Sequence-specific 1H NMR assignments and secondary structure of the streptococcal protein G B2-domain.

Two-dimensional NMR spectroscopy has been used to obtain sequence-specific 1H NMR assignments for the IgG-binding B2-domain of streptococcal protein G. Secondary structure elements were identified from analysis of characteristic backbone-backbone NOE patterns and amide proton exchange data. The B2-domain contains a four-stranded beta-sheet region in which the two inner strands form a parallel beta-sheet with each other and antiparallel beta-sheets with the outer strands. The outer strands are connected via a 16-residue alpha-helix and short loops on both ends of the helix. The alpha-helix and beta-sheet structures contain well-defined polar and apolar sides, and numerous long-range NOEs from the apolar helix to apolar sheet regions were used to derive a model for the global fold of the B2-domain. While the overall fold is similar to that obtained for B1-type domains, differences in amide proton exchange rates and hydrophobic packing are observed.     

Two-dimensional crystallization on lipid monolayers and three-dimensional structure of sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.     

Sequence-specific 1H NMR assignments and determination of the secondary structure for the activation domain isolated from pancreatic procarboxypeptidase B

Nearly complete sequence-specific 1H NMR assignments are presented for amino acid residues 3-81 in the 81-residue globular activation domain of porcine pancreatic procarboxypeptidase B isolated after limited tryptic proteolysis of the zymogen. These resonance assignments are consistent with the chemically determined amino acid sequence. Regular secondary structure elements were identified from nuclear Overhauser effects and the sequence locations of slowly exchanging backbone amide protons. The molecule contains two alpha-helices, including residues 20-30 and approximately residues 58-72, and a three-stranded antiparallel beta-sheet with the individual strands extending approximately from 12 to 17, 50 to 55, and 75 to 77. The identification of these secondary structures and a preliminary analysis of additional long-range NOE distance constraints show that isolated activation domain B forms a stable structure with the typical traits of a globular protein. The data presented here are the basis for the determination of the complete three-dimensional structure of activation domain B, which is currently in progress.     

Sizing the radius of the pore formed in erythrocytes and lipid vesicles by the toxin sticholysin I from the sea anemone Stichodactyla helianthus

Tamoxifen (tmx) is a non-steroidal triphenylethylene derivative that is predominantly known as a competitive antagonist at the estrogen receptor and is used in the treatment of breast cancer. Recent studies suggest that tamoxifen is also beneficial in the treatment of brain metastases and primary brain tumors. Tmx accumulates in brain and its concentration can be up to 46-fold higher than in serum. Therefore, astrocytes may be exposed to tmx in vivo. We use the whole-cell patch-clamp technique to examine the effects of tmx on voltage-dependent cation currents in rat cortical cultures. Using biophysical and pharmacological methods, we isolate sustained and transient outward potassium currents (I _KS and I _KT , respectively), inwardly rectifying potassium currents (I _KIR ), and transient inward sodium currents (I _Na ). We show that that TTX-sensitive I _Na is completely inhibited by 10 μm tmx within 5 min. Similarly, tmx blocks I _KS , but does not inhibit I _KT or I _KIR at these concentrations. Tmx effects are irreversible with 10 min wash. Interestingly, the currents sensitive to tmx are important in growth control of glial cells (MacFarlane & Sontheimer, 1997). Therefore, we examine cytotoxic and proliferative effects of tmx. Tmx (10 μm) is not cytotoxic as judged by trypan blue exclusion. However, incubation with tmx significantly reduces cell proliferation as examined by ³[H]-thymidine uptake. Received: 12 October 2000/Revised: 12 February 2001     

Overproduction in Escherichia coli and purification of the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus

The cDNA coding for the cytolytic toxins sticholysin I and sticholysin II from the sea anemone Stichodactyla helianthus has been isolated, cloned in pUC18, and sequenced. A 6His-tagged version of sticholysin II has been overproduced in Escherichia coli and purified to homogeneity in milligram amounts. Conformational and functional analyses of recombinant sticholysin II do not reveal any significant difference when compared to the natural cytolysin.     

Immunohistochemical targeting of sea anemone cytolysins on tentacles, mesenteric filaments and isolated nematocysts of Stichodactyla helianthus

The toxicity of biomolecules obtained from sea anemones in vitro does not necessarily justify their function as toxins in the physiology of the anemone. That is why anatomical and physiological considerations must be taken into account in order to define their physiological role in the organism. In this work, antibodies generated to Sticholysin II, a cytolysin produced by the Caribbean Sea anemone Stichodactyla helianthus, are used as specific markers to explore the sites of production and storage of the cytolysin in the sea anemone. The immunoperoxidase staining developed gave specific dark-brown staining in tentacles and mesenteric filaments as well as in basitrichous nematocysts isolated from tentacles of S. helianthus. These results support the role of these proteins as toxins in the physiology of the anemone, especially in functions such as in predation, defense and digestion.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Sequential 1H NMR assignments and secondary structure of aponeocarzinostatin in solution

Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.     

1H and 15N NMR resonance assignments and secondary structure of titin type I domains

Titin/connectin is a giant muscle protein with a highly modular architecture consisting ofmultiple repeats of two sequence motifs, named type I and type II. Type I modules have beensuggested to be intracellular members of the fibronectin type III (Fn3) domain family. Alongthe titin sequence they are exclusively present in the region of the molecule located in thesarcomere A-band. This region has been shown to interact with myosin and C-protein. Oneof the most noticeable features of type I modules is that they are particularly rich insemiconserved prolines, since these residues account for about 8% of their sequence. We havedetermined the secondary structure of a representative type I domain (A71) by 15N and 1HNMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting ofa three- and a four-stranded -sheet. When the two sheets are placed on top of each other toform the -sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the sameside of the molecule and form an exposed hydrophobic patch. This suggests that thesemiconserved prolines might be relevant for the function of type I modules, providing asurface for binding to other A-band proteins. The secondary structure of A71 was structurallyaligned to other extracellular Fn3 modules of known 3D structure. The alignment shows thattitin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.     

Sequence-specific 1H NMR assignment and secondary structure of neuropeptide Y in aqueous solution

Sequence-specific assignment of the 1H NMR spectrum of the 36 amino acid polypeptide porcine neuropeptide Y (pNPY) at pH 3.1 is reported. It was achieved by use of standard two-dimensional techniques and by a combination of the sequential and main-chain-directed assignment strategies. The secondary structure was derived from inspection of the nuclear Overhauser spectra, slow hydrogen-deuterium exchange effects, chemical shifts of main-chain HA resonances, and coupling constants. These studies indicate that the C-terminal segment (residues 11-36) folds into an amphiphilic alpha-helix; the N-terminal segment, containing three prolines in both cis and trans conformations, assumes no regular structure. CD studies of pNPY at pH 3.1 and 7.4 show an increase in ordered structure at neutral pH. The difference spectrum, however, is typical of an alpha-helix and suggests a stabilization of residues 11-36, possibly via Maxfield-Scheraga pair interactions involving side-chain residues. This is supported by a comparison of the one-dimensional 1H NMR spectra of pNPY at pH 3.1 and 7.4, where no remarkable differences are observed.