Synthesis and conformational analysis of apamin analogues with natural and non-natural cystine/selenocystine connectivities.

By replacing two cysteine residues in apamin with selenocysteine, the three possible isomers related to the side-chain connectivities of a bis-cystinyl-peptide were synthesized in regioselective manner exploiting the low redox potential of the diselenide bond. Nuclear magnetic resonance conformational analysis of monoselenocystine analogue apamin with the natural diselenide/disulfide network confirmed the highly isomorphous character of the sulfur replacement with selenium despite its slightly larger atomic radius and increased bond lengths. The comparative conformational analysis of the apamin analogues containing the non-natural side-chain links with wild type apamin clearly revealed retention of the main structural fold and thus the high propensity of these small molecules to adopt the secondary structure elements present in natural apamin. These findings offered interesting hints for a better understanding of the oxidative refolding pathway of the bis-cystinyl peptide that leads exclusively to the correct natural isomer.

The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized alpha-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.

Isomorphous replacement of cystine with selenocystine in endothelin: oxidative refolding, biological and conformational properties of [Sec3,Sec11,Nle7]-endothelin-1.

Air re-oxidation of fully reduced human endothelin-1 under optimized conditions yields the natural isomer with parallel disulfide bridges and the non-natural isomer with crossed disulfide bridges at a ratio of 3:1. In view of the recently determined highly reducing redox potential of selenocysteine (-381 mV) in peptides, the half-cystine residues Cys3 and Cys11 of the natural isomer of endothelin-1 were replaced by selenocysteine. Taking advantage of the high stability of the diselenide group toward reducing agents for disulfides a regioselective disulfide bridging of the second cysteine pair allowed for straightforward preparation of the [Sec3,Sec11, Nle7]-endothelin-1. NMR structural analysis showed conformational preferences of this endothelin analog that were identical to those of the natural hormone. Similarly, the bioactivity data confirmed that replacement of cysteine residues with selenocysteine was without detectable effect on receptor recognition and signal transduction. Both findings strongly support that the exchange of sulfur against selenium produces a fully isomorphous molecule as recently observed for similar exchanges at the level of methionine residues in proteins. Moreover, oxidative refolding of the fully reduced [Sec3,Sec11,Nle7]-endothelin-1 fulfilled the expectation that the redox potential of the selenocysteines would dictate quantitative formation of the natural isomer. These results suggest that the selenocysteine approach, besides offering an interesting chemical tool for induction of correct oxidative folding of multiple cysteine-containing peptides, should even allow for the preparation of non-natural isomers and thus for studying conformational preferences of folding intermediates in peptides and proteins.

Cyclodextrins as templates for the presentation of protease inhibitors.

Mono(6-succinylamido-6-deoxy)-beta-cyclodextrin was synthesized by classical carbohydrate chemistry and used as a template mono-functionalized with the linear, fully flexible 4C-spacer carboxylate for covalent linkage of the calpain inhibitor leucyl-leucyl-norleucinal. Spectroscopic analyses of the conjugate do not support a self-inclusion of part of the hydrophobic peptide tail, but confirm its intra- or intermolecular interaction with the template moiety that leads to full water solubility. The inhibitory potency of the beta-cyclodextrin/peptide aldehyde construct was compared with that of the parent Ac-Leu-Leu-Nle-H against cathepsin B and calpain. Despite the large size of the template the inhibition of cathepsin B was only slightly reduced in full agreement with the X-ray structure of this enzyme which shows full accessibility of the S-subsites. For this enzyme the 4C-spacer is apparently sufficient to guarantee optimal interaction of the peptide tail with the binding cleft. Conversely, for mu-calpain a significantly decreased inhibitory potency was obtained with the conjugate suggesting steric interference of the template in the binding process. These results show that the beneficial properties of the cyclodextrin template can be retained in conjugates with bioactive peptides if attention is paid to optimize in each case the size and nature of the spacer for optimal recognition of the grafted biomolecule.

Oxidative folding of cystine-rich peptides vs regioselective cysteine pairing strategies.

The methodology of regioselective cysteine pairings in synthetic multiple-cystine peptides has progressed in the past years to an efficiency that allows for at least three specific inter- and intrachain disulfide bridgings. Conformational studies on various multiple-cystine peptides like hormones, protease inhibitors, and toxins revealed that these bioactive peptides, generated by posttranslational processing of precursor proteins, are folded into miniprotein-like compact globular structures of remarkable stability. This strongly suggests protein domain or subdomain properties of these families of peptides, and thus sufficient sequence-encoded information for correct oxidative refolding under appropriate experimental conditions. From intensive research on the mechanisms and pathways of oxidative refolding of proteins in vivo and in vitro, the efficient methods have emerged for simulating nature in the regeneration of native folds not only for intact proteins, but also for protein domains and subdomains. In fact, the results obtained in the oxidative folding of excised protein fragments and of relatively low mass products of posttranslational processings show that this procedure is indeed a simple way of preparing peptides with several disulfide bonds, if optimization of reaction conditions is performed in terms of redox buffer, temperature, and additives capable of disrupting aggregates and of stabilizing nascent secondary structures. Moreover, with increased knowledge about stable, small natural cystine frameworks, their use instead of artificial templates should facilitate engineering of synthetic miniproteins with specific conformation and tailored functions.

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation.

Bis(cysteinyl)octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10(-3) M concentration was found to be very similar, but opposite to that of the Kox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of beta-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (approximately 90%). Similarly, operating under denaturing conditions, i.e., in 6 M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of beta-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence.

Molecular structure of the cyanobacterial tumor-promoting microcystins.

The three-dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat-like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.

Identification and characterization of microcystin-LY from Microcystis aeruginosa (strain 298).

Two toxins, a main component A and a minor component B, were isolated from the freshwater cyanobacterium Microcystis aeruginosa (strain 298) and characterized in their chemical structure by amino-acid analysis, configurational analysis, by FAB-MS and 1H-NMR spectroscopy. The acid hydrolysate yielded for toxin A as constituent amino acids D-Ala, L-Leu, D-Glu, erythro-D-beta-Me-Asp and L-Arg, and for toxin B the amino acids D-Ala, L-Leu, D-Glu, erythro-D-beta-Me-Asp and L-Tyr. 1D and 2D 1H-NMR spectroscopy of the toxins A and B in DMSO-d6 allowed to characterize them as cyclic heptapeptides containing both the unusual beta-amino acid Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) and N-methyldehydroalanine (Mdha) as additional constituent residues. Toxin A was found to correspond to the known and structurally well characterized microcystin-LR and toxin B to microsystin-LY. The presence of this variant has already been proposed, but its primary structure could be confirmed in this study.

Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases.

The active sites of thiol-protein oxidoreductases consist of the characteristic Cys-X-X-Cys motif, and the redox potentials of these enzymes reflect the propensity of the bis(cysteinyl) sequence portion for disulfide loop formation. Thereby, as is known from comparing the three-dimensional (3D) structures of thioredoxin and glutaredoxin in the reduced and oxidized state, reduction of the disulfide bond is accompanied by minimal perturbation of the backbone folding of the active sites. In order to estimate the sequence-dependent intrinsic free energy of formation of the active-site disulfide loops in oxidoreductases, synthetic fragments corresponding to the sequences 31-38, 10-17, 134-141, and 34-41 of thioredoxin, glutaredoxin, thioredoxin reductase, and protein disulfide isomerase (PDI), respectively, were analyzed for their tendency to form 14-membered rings. For this purpose thiol/disulfide exchange experiments, with glutathione as reference redox pair, were performed on the bis(cysteinyl) octapeptides. As the free energy of ring closure of linear peptides consists mainly of the free energy of formation of the disulfide loop with a defined geometry from a statistical ensemble of conformations of the bis(cysteinyl) peptides, the observed differences in the equilibrium constants, although relatively small (within a factor 10), suggest that sequence-dependent information for loop formation is retained in the excised active-site fragments. These inherent redox potentials are, however, significantly affected and/or amplified in the native proteins by the conformational restraints imposed by the "structural domains" on the "functional domains".