Synthesis and secondary structural studies of penta(acetyl-Hmb)A beta(1-40).

The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native A beta(1-40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of A beta(1-40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)A beta(1-40) was found to adopt both alpha-helix and intermolecular beta-sheet conformations. In 100% TFE a mixed alpha-helix/random coil structure was formed by the protected peptide indicating reduced alpha-helical propensity relative to A beta(1-40). The protected peptide formed beta-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native A beta(1-40), penta(acetyl-Hmb)A beta(1-40) did not form large aggregate species.     

Immunization with a peptide having both T cell and conformationally restricted B cell epitopes elicits neutralizing antisera against a snake neurotoxin.

We have synthesized a free peptide capable of eliciting antibodies that neutralize toxin alpha from Naja nigricollis, a protein that binds specifically to the acetylcholine nicotinic receptor. Of the five tested fragments that encompassed the whole toxin sequence, only fragment 24-41 stimulated T cells from BALB/c mice primed with the whole toxin and conversely, only T cells from mice primed with fragment 24-41 could be stimulated by both the toxin and priming peptide. No other peptides had such properties, indicating that only fragment 24-41 possessed T determinant(s) in BALB/c mice (H-2d haplotype). In agreement with the current view that B cell proliferation requires specific T cell stimulation, only fragment 24-41 elicited an antibody response. However, the antipeptide antisera failed to bind to the native toxin and thereby to neutralize it. Instead, it recognized an unfolded form of the toxin. The peptide 24-41 was then made cyclic. A circular dichroism analysis revealed that, in organic solvent, this peptide had a tendency to adopt a beta-sheet structure, as in the folded toxin, whereas the linear peptide adopted an helical structure. The cyclic peptide not only remained T stimulating but elicited antisera that recognized and neutralized the native toxin. Furthermore, the antisera cross-reacted with several toxin variants. Our data show, therefore, that it is possible to give an appropriate B cell specificity directly to a T cell-stimulating peptide, an approach that may be of value for the design of synthetic vaccines.     

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.     

Structural elements regulating amyloidogenesis: a cholinesterase model system

Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE(586-599), through the effect of single point mutations on beta-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE(586-599) was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE(586-599) assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high beta-strand propensity, for the conformational transition to beta-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to beta-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE(586-599) surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE(586-599) assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-pi, SH-aromatic, metal chelation and polar-polar) would maintain the beta-sheets together. We also propose that the stacking between the strands in the beta-sheets along the fiber axis could be stabilized through pi-pi interactions and metal chelation. The dissection of the specific molecular recognition driving AChE(586-599) amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases.     

Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin

Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.     

Probing local secondary structure by fluorescence: time-resolved and circular dichroism studies of highly purified neurotoxins.

The relationship between beta-sheet secondary structure and intrinsic tryptophan fluorescence parameters of erabutoxin b, alpha-cobratoxin, and alpha-bungarotoxin were examined. Nuclear magnetic resonance and x-ray crystallography have shown that these neurotoxins have comparable beta-sheet, beta-turn, and random coil secondary structures. Each toxin contains a single tryptophan (Trp) residue within its beta-sheet. The time-resolved fluorescence properties of native erabutoxin b and alpha-cobratoxin are best described by triple exponential decay kinetics, whereas native alpha-bungarotoxin exhibits more than four lifetimes. The disulphide bonds of each toxin were reduced to facilitate carboxymethylation and amidocarboxymethylation. The two different toxin derivatives of all three neurotoxins displayed triple exponential decay kinetics and were completely denatured as evidenced by circular dichroism (random coil). The concentration (c) values of the three fluorescence decay times (time-resolved fluorescence spectroscopy (TRFS)) were dramatically different from those of the native toxins. Each neurotoxin, treated with different concentrations of guanidinium hydrochloride (GuHCl), was studied both by circular dichroism and TRFS. Disappearance of the beta-sheet secondary structural features with increasing concentrations of GuHCl was accompanied by a shift in the relative contribution (c value) of each fluorescence decay time (TRFS). It was found that certain disulphide residues confer added stability to the beta-sheet secondary structure of these neurotoxins and that the center of the beta-sheet is last to unfold. These titrations show that Trp can be used as a very localized probe of secondary structure.     

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation,but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.     

A peptide, containing the universal antigenic determinant of tryptophanyl-tRNA-synthetase]

Among clostripain hydrolysate peptides of beef pancreas tryptophanyl-tRNA synthetase the peptide Ile-Ser-Phe-Pro-Ala-Ile-Asn-Gln-Phe-Ala-Ala-Pro-Ser-Gln-Phe-Ser-Ile-Arg was revealed which contains the continuous antigenic determinant for monoclonal antibody Am1. This antibody specifically cross-reacts with tryptophanyl-tRNA synthetases of procaryotes, eucaryotes and archebacteriae. The synthetic peptide with identical amino acid sequence plus N-terminal Arg residue (S-peptide), being immobilized on enzyme immunoassay (EIA) microtitration plate, also binds with Am1. Am1 affinity constant (M-1) measured by non-competitive EIA was (3.0 +/- 0.3).10(7) for S peptide and (1.4 +/- 0.3).10(9) for the native enzyme. The sequence of immunoreactive peptide adopts with high probability the secondary structure including beta-turn(s) and antiparallel beta-sheet composed of inverted repeats. At the same time, the analysis of circular dichroism spectrum (in the far UV) of the peptide dissolved in water comes closest to 16% beta-turn and only 8% beta-sheet. The binding of Am1 with peptide was not observed in aqueous solution.     

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

Interaction of Synthetic Peptides from Fasciculin with Acetylcholinesterase

Fasciculins (Fas) are three-looped polypeptides isolated from mamba venom which exert their toxic action by inhibiting noncompetitively acetylcholinesterase (AChE). A peptide (Fas-D) encompassing the first loop sequence was synthesized and characterized chemically, structurally, and functionally. Fas-D possesses an intramolecular disulfide bridge, present in the native toxin. Circular dichroism (CD) indicated the existence of 21.8% -sheet content and 24.2% -turn in this peptide, compatible with crystallographic data of the native toxin. The peptide showed only low partial AChE inhibition at submillimolar concentrations, much lower than that observed with Fas and a peptide (Fas-B) encompassing the second loop sequence. The simultaneous presence of Fas-D and Fas-B produced an additive inhibitory effect on AChE activity; calculated K _i and K _i values (7.3 ± 2.4 M and 10.0 ± 1.8 M, respectively) were not significantly different, thus indicating noncompetitive inhibition. These results are consistent with site-directed mutagenesis studies and analysis of the crystal structure of the Fas–AChE complex, which indicate that residues from loops I and II contribute to Fas binding to the enzyme.     

Interaction of peptide boronic acids with elastase: circular dichroism studies

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.     

Synthesis and structural characterization of charybdotoxin, a potent peptidyl inhibitor of the high conductance Ca2(+)-activated K+ channel.

Charybdotoxin (ChTX), a potent inhibitor of the high conductance Ca2(+)-activated K+ channel (PK,Ca) is a highly basic peptide isolated from venom of the scorpion Leiurus quinquestriatus hebraeus, whose primary structure has been determined (Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczorowski, G. J., and Garcia, M. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3329-3333). The synthesis of this peptide using continuous flow solid phase fluorenylmethyloxycarbonyl-pentafluorophenyl ester methodology has now been achieved. The 1-37-amino acid hexasulfhydryl peptide oxidizes readily to give the tricyclic disulfide structure in good yield. This folded synthetic material is identical to native toxin based on three criteria: co-migration with ChTX on reversed phase high performance liquid chromatography (HPLC); competitive inhibition of 125I-labeled monoiodotyrosine charybdotoxin binding to bovine aortic sarcolemmal membrane vesicles with a Ki (10 pM) identical to that of native toxin; blockade of PK,Ca activity in excised outside-out patches from bovine aortic smooth muscle with the potency and inhibitory properties characteristic of ChTX (i.e. appearance of silent periods interdispersed with normal bursts of channel activity in single channel recordings). Selective enzymatic digestion of native or synthetic ChTX by simultaneous exposure to chymotrypsin and trypsin yields identical reversed phase HPLC profiles. Analysis of the sequence and amino acid composition of the resulting fragments defines a disulfide bond arrangement (Cys7-Cys28, Cys13-Cys33, Cys17-Cys35) which differs from that previously suggested. This configuration predicts a highly folded tertiary structure for ChTX which, together with observations from electrophysiological and binding experiments, suggests a possible mechanism by which ChTX interacts with PK,Ca to block channel function.     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

The role of Phe in the formation of well-ordered oligomers of amyloidogenic hexapeptide (NFGAIL) observed in molecular dynamics simulations with explicit solvent

We observed fast aggregation of partially ordered oligomers in an earlier simulation study of an amyloidogenic hexapeptide NFGAIL. In this work, the nucleation of highly ordered oligomers was further investigated by a combined total of 960 ns molecular dynamics simulations with explicit solvent on NFGAIL and its nonamyloidogenic mutant NAGAIL. In these simulations, four dimer subunits that each was constrained by harmonic forces as a two-strand beta-sheet were used to enhance the rate of formation. It was found that a critical role played by the aromatic residue Phe was to direct the stacking of beta-sheets to form ordered multilayer aggregates. We also found that many molecular arrangements of the peptide satisfied the "cross-beta-structure", a hallmark of amyloid fibrils. The tendency for the peptide to form either parallel or antiparallel beta-sheet was comparable, as was the tendency for the beta-sheets to stack either in parallel or antiparallel orientation. Overall, approximately 85% of the native hexapeptide formed octamers. The fact that only 8% of the octamers were well-ordered species suggests that the dissociation of the disordered oligomers be the rate-limiting step in the formation of highly ordered oligomers. Among the well-ordered subunit pairs, about half was formed by the beta-sheet extension along the main-chain hydrogen-bond direction, whereas the other half was formed by the beta-sheet stacking. Hence, a delicate balance between intersheet and intrasheet interactions appeared to be crucial in the formation of a highly ordered nucleus of amyloid fibrils. The disordered oligomers were mainly stabilized by nonspecific hydrophobic interactions, whereas the well-ordered oligomers were further stabilized by cross-strand hydrogen bonds and favorable side-chain stacking.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Amphiphilic Detergent-Soluble Acetylcholinesterase from Torpedo marmorata: Characterization and Conversion by Proteolysis to a Hydrophilic Form

The membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X-100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent-soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X-100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent-solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent-soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.     

Transformation of the amyloidogenic peptide amylin(20-29) into its corresponding peptoid and retropeptoid: access to both an amyloid inhibitor and template for self-assembled supramolecular tapes

The highly amyloidogenic peptide sequence of amylin(20-29) was transformed into its corresponding peptoid and retropeptoid sequences to design a novel class of beta-sheet breaker peptides as amyloid inhibitors. This report describes the synthesis of the chiral peptoid building block of L-isoleucine, the solid phase synthesis of the peptoid and retropeptoid sequences of amylin(20-29), and the structural analysis of these amylin derivatives in solution by infrared spectroscopy, circular dichroism, and transmission electron microscopy. It was found that the peptoid sequence did not form amyloid fibrils or any other secondary structures and was able to inhibit amyloid formation of native amylin(20-29). Although the retropeptoid did not form amyloid fibrils it had only modest amyloid inhibitor properties since supramolecular tapes were formed.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% beta-sheet, 28% aggregated beta-strands, 10% alpha-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of alpha-helices, turns and loops, and beta-structures did not change, however, the 1636cm(-1) beta-sheet band increased from 18% to 31% at the expense of the 1680cm(-1) beta-sheet structure. Spectral analysis of the amide I band showed that the alpha-helical component was oriented with at 41 degrees to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.