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. © 1996 John Wiley & Sons, Inc.

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The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfidecoupled folding into the cystine-stabilized a-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 diselenide0disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.

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".

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