Differential involvement of disulfide bridges on the folding of a scorpion toxin

Leiurotoxin I is a neurotoxin, blocker of Ca²⁺-activated apamin-sensitive K⁺ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys³-Cys²¹, Cys⁸-Cys²⁶ and Cys¹²-Cys²⁸. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by ¹H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys³-Cys²¹ and a loss of organized structure for another analog lacking disulfide bridge Cys¹²-Cys²⁸. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys¹²-Cys²⁸ is essential for the folding process. In contrast, the lack of disulfide bridge Cys³-Cys²¹ does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.     

Attempt to map the receptor binding sites of human follicle‐stimulating hormone using disulfide peptides of its β‐subunit indicates major involvement of the regions around disulfide bonds Cys28–Cys82 and Cys32–Cys84 in receptor binding of the hormone

Abstract: Follicle‐stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The α‐subunit which is common within a species and the β‐subunit which is hormone‐specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross‐linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSHβ in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSHβ were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [¹²⁵I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys²⁸–Cys⁸² and Cys³²–Cys⁸⁴ were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys²⁸–Cys⁸² and Cys³²–Cys⁸⁴ in receptor binding of the hormone. The studies also suggest the involvement of βL2 and βL3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.     

Combined use of ESI‐MS and UV diode‐array detection for localization of disulfide bonds in proteins: application to an α‐l‐fucosidase of pea

Abstract: A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10–30 kDa). The method combines the use of high‐performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC‐ESI‐MS) and HPLC with UV diode‐array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in α‐l ‐fucosidase from pea. The two disulfide bonds were located between residues Cys⁶⁴ and Cys¹⁰⁹ and between Cys¹⁶² and Cys¹⁶⁹, while Cys¹²⁷ was free.     

Paralytic activity of (des-Glu1)conotoxin GI analogs in the mouse diaphragm

A series of 20 peptide analogs of (des-Glu¹) conotoxin GI were prepared by solid phase synthesis. The peptides were tested for their abilities to inhibit contractions in the mouse-diaphragm-with-phrenic-nerve assay. (Des-Glu¹) conotoxin has an IC₅₀, of 2.7 × 10^?7 M in this assay. Results from this assay show that total loss of paralytic activity occurs when Pro is replaced by Gly, Tyr by D-Tyr, or Gly by D-Phe. In most cases loss or change in length of one of the disulfide rings eliminates paralytic activity except with compound 17, which is weakly active, IC₅₀= 7.0 × 10^?5 M. Replacement of the Cys¹-Cys⁶ disulfide bond with an amide bond (compound 9) greatly lowers paralytic activity, IC₅, = 3.7 × 10^?5 M.     

A comparison of folding techniques in the chemical synthesis of the epidermal growth factor‐like domain in neu differentiation factor α/β

Abstract: The 52‐residue α/β chimera of the epidermal growth factor‐like domain in neu differentiation factor (NDF_eα/β) has been synthesized and folded to form a three disulfide bridge (Cys¹⁸²–Cys¹⁹⁶, Cys¹⁹⁰–Cys²¹⁰, Cys²¹²–Cys²²¹) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single‐step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one‐step approach may not always provide the desired disulfide pairing. Here, we compare the single‐step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys¹⁸² to Cys¹⁹⁶, Cys¹⁹⁰ to Cys²¹⁰ and Cys²¹² to Cys^221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP‐HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via I₂ oxidation. Only the peptide having the Cys¹⁸²–Cys¹⁹⁶ pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys¹⁹⁰–Cys²¹⁰/Cys²¹²–Cys²²¹) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large‐scale syntheses of NDF_eα/β and other novel EGF‐like peptides.     

Maurotoxin and the Kv1.1 channel: voltage‐dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31–Cys34

Abstract: Maurotoxin (MTX) is a 34‐amino acid polypeptide cross‐linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half‐cystine pairings at Cys³–Cys²⁴, Cys⁹–Cys²⁹, Cys¹³–Cys¹⁹ and Cys³¹–Cys^34. This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C‐terminal ‘14‐membered disulfide ring’ (i.e. cyclic domain 31–34), We therefore studied structure–activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C‐terminus {i.e. MTX (1–29), [Abu_31,34]‐MTX and [Cys_31,34, Tyr₃₂]_d ‐MTX} or mimicking the cyclic C–terminal domain [i.e. MTX (31–34)]. Unexpectedly, the absence of a disulfide bridge Cys³¹–Cys³⁴ in [Abu _31,34]‐MTX and MTX (1–29) resulted in MTX‐unrelated half‐cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX (31–34) was also biologically inactive. [Cys_31,34, Tyr₃₂]_d ‐MTX, which had a ‘native’, MTX‐related, disulfide bridge organization, but a d ‐residue‐induced reorientation of the C–terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide‐induced Kv1.1 blockage was voltage‐dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on‐rate changes in ligand binding. Thus, the cyclic C–terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Controlled syntheses of natural and disulfide-mispaired regioisomers ofα-conotoxin SI*

Abstract: Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of α-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two ‘interlocking’ disulfide bridges connecting Cys²–Cys⁷ and Cys³–Cys¹³ (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys²–Cys¹³ and Cys³–Cys⁷ (2/13 & 3/7, ‘nested’) and Cys²–Cys³ and Cys⁷–Cys¹³ (2/3 & 7/13, ‘discrete’), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation ofS-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Combined use of NMR,distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)-Cys2, D(L)-Cys5]enkephalin

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr¹-Cys²-Gly³ Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr¹-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr¹-D-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyr^l-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.     

Three‐dimensional solution structure of a disulfide bond isomer of the human insulin‐like growth factor‐I

Abstract: The solution structure of a disulfide bond isomer of human insulin‐like growth factor‐I (IGF‐I) was determined using homonuclear NMR methods. A total of 292 interatomic distance constraints, including 12 related to the disulfide bridges, was used in the distance geometry calculations. The determined structures contain two helical rods corresponding to the sequence regions, Ala⁸–Cys¹⁸ and Leu⁵⁴–Cys⁶¹. Comparison with the previously determined structure of native human IGF‐I revealed partial correspondence of the secondary structure (helices I: Ala⁸–Cys¹⁸ and helices III: Leu⁵⁴–Cys⁶¹) and internal packing. Helix II in native human IGF‐I (residues Gly⁴²–Cys⁴⁸) is disrupted in the isomer. A similar relationship has been described between the structure of native insulin and a homologous disulfide isomer, suggesting that these alternative folds represent general features of insulin‐like sequences. In each case the precision of the distance geometry ensemble is low due in part to resonance broadening and a paucity of NOEs relative to other globular proteins of this size. These observations suggest that tertiary structure of the isomer is not highly ordered. Comparison of the biological activities of native and the disulfide bond isomer of human IGF‐I highlight the importance of Tyr²⁴, Phe²⁵, Phe⁴⁹–Cys⁵² and Phe¹⁶ in binding to the IGF‐I receptor or specific IGFBPs. The relationship of this proposed receptor‐binding surface of human IGF‐I to those of insulin is discussed.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

Characterization of epitope regions of thyrotropin β-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach

Abstract: This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin β-subunit, bTSHβ. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHβ developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised β-turn regions of loop L1[bTSHβ(14-20)] and loop L3[bTSHβ(65-72)] held in close proximity by a bis-β-alanine linker and the disulfide bond bTSHβ[Cys¹⁶-Cys⁶⁷]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHβ[¹²⁵I-bTSHβ] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHβ that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of ¹²⁵I-bTSHβ to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHβ-epitope recognized by mAb299 involves contributions from amino residues from the β-turn regions of the L1 and L3 loops of TSHβ, and that these loop regions flank part of the receptor binding site of the bTSH β-subunit.     

Relationship between temporary inhibition and structure of disulfide‐linkage analogs of marinostatin,a natural ester‐linked protein protease inhibitor

Abstract: A 12‐residue marinostatin [MST(1–12): ¹FATMRYPSDSDE¹²] which contains two ester linkages of Thr³–Asp⁹ and Ser⁸–Asp¹¹ strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K_i value of 1SS(C³–C⁹) (¹FACMRYPSCSDE¹²), which has a single disulfide linkage of Cys³–Cys⁹ was comparable with those of MST(1–12) and MST‐2SS (¹FACMRYPCCSCE¹²), a doubly linked analog of Cys³–Cys⁹ and Cys⁸–Cys¹¹. However, 1SS(C³–C⁹) and MST‐2SS showed temporary inhibition, but not MST(1–12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. ¹H NMR study showed that 1SS(C³–C⁹) has two conformations, which contain a cis‐ (70%) or trans‐ (30%) Pro residue, while MST‐2SS as well as MST(1–12) takes a single conformation containing only a cis‐Pro residue. Hydrogen–deuterium exchange rate of the Arg⁵ (P1′) NH proton of the MST analogs was about 100 times faster than that of MST(1–12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis‐conformation of the Pro⁷ residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease‐inhibitor complex.     

Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector

The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a “protein” or a “peptide” program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after performic oxidation, and by partial labeling of the half cystine residues with [¹⁴C]-iodoacetic acid and determining the specific radioactivities of the S-[¹⁴C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys¹² in mammal toxin II is replaced by Cys³⁸ in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.     

Synthesis and biological characterisation of a series of iberiotoxin analogues

We report here the synthesis of iberiotoxin (IbTX), a 37-amino acid peptide containing three disulfide bridges, and a series of mono-looped analogues. All syntheses were conducted using Fmoc chemistry. Synthesis of IbTX gave a product which was indistinguishable from a reference sample in both its physicochemical properties and its biological activity. A series of three mono-looped analogues, in which four of the six cysteines were replaced by alanine, were synthesised to give [Ala^7,13,28,33]-IbTX, [Ala^13,17,33,35]-IbTX and [Ala^7,17,28,35]-IbTX. Oxidation of the linear form of [Ala^7,17,28,35]-IbTX to form the Cys¹³ to Cys³³ disulfide bridge proceeded more slowly than that of the other two analogues. None of these analogues was biologically active, indicating that no single loop is the mediator of channel blocking activity.     

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective Somatostatin derivatives

Three cyclic disulfide analogs related to somatostatin, d -Phe¹ -Cys²-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-Xxx⁷-Thr⁸NH₂ (where Xxx =l -Pen 1; l -Cys 3; or d -Pen 4) were examined in DMSO-d₆ by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a β II turn solution conformation for the core tetrapeptide-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.     

Cyclic melanotropins

The highly potent cyclic analogue of α-MSH, Ac-[Cys⁴, Cys¹⁰]-α-MSH_4–13-NH₂, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa⁴, Cys¹⁰]-α-MSH_4–13-NH₂ resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys⁴, Cys¹⁰]-α-MSH_4–13-NH₂ led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa⁴, Cys¹⁰]-α-MSH_4–13-NH², resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the ring by one methylene group to give Ac-[Hcy⁴, Cys¹⁰]-α-MSH_4–13-NH₂ led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.     

Chemical synthesis and structure–activity relationships of Ts κ, a novel scorpion toxin acting on apamin-sensitive SK channel

Abstract: Tityus kappa (Ts κ), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts κ was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs κ, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC₅₀ = 3 nm ). The sTs κ was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD₅₀ = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs κ; they were between Cys₇–Cys₂₈, Cys₁₃–Cys₃₃ and Cys₁₇–Cys₃₅, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts κ (i.e. Arg₆, Arg₉, Lys₁₈, Lys₁₉) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by ¹H-NMR, suggest that Arg₆, and to a lesser extent Arg₉, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the α-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the α-helix.     

Cystine peptides Antiparallel β-sheet conformation of the cyclic biscystine pep tide [Boc-Cys-Ala-Cys-NHCH3]2

The crystal structure analysis of the cyclic biscystine peptide [Boc-Cys¹-Ala²-Cys³-NHCH₃]₂ with two disulfide bridges confirms the antiparallel β-sheet conformation for the molecule as proposed for the conformation in solution. The molecule has exact twofold rotation symmetry. The 22-membered ring contains two transannular NH ? OC hydrogen bonds and two additional NH ? OC bonds are formed at both ends of the molecule between the terminal (CH₃)₃COCO and NHCH₃ groups. The antiparallel peptide strands are distorted from a regularly pleated sheet, caused mainly by the L-Ala residue in which φ=– 155° and ψ= 162°. In the disulfide bridge C^α (1)-C^β (1)-S(1)-(3′)-C^β(3′)-C^α(3′), S—S = 2.030 Å, angles C^β SS = 107° and 105°, and the torsional angles are –49, –104, +99, –81, –61°, respectively. The biscystine peptide crystallizes in space group C2 with a = 14.555(2) Å, b = 10.854(2) Å, c = 16.512(2)Å, and β= 101.34(1) with one-half formula unit of C₃₀H₅₂N₈O₁₀S₄· 2(CH₃)₂SO per asymmetric unit. Least-squares refinement of 1375 reflections observed with |F| > 3σ(F) yielded an R factor of 7.2%.