Oxidative folding of cystine‐rich peptides vs regioselective cysteine pairing strategies

Abstract

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.

The CXC Motif: A Functional Mimic of Protein Disulfide Isomerase

“…CXC peptides can form 11-membered disulfide-bonded rings with E°′ values that are typically 30-40 mV (1.4-1.8 kcal/mol) less stable than those of CXXC peptides (56,53). The structural basis for the relative instability of CXC disulfide-bonded rings relative to CXXC disulfide-bonded rings is not entirely clear.…”

Diselenides as universal oxidative folding catalysts of diverse proteins

“…The active sites of these oxidoreductases have inspired the design of smallmolecule catalysts that can serve as alternatives to glutathione during in vitro applications. For example, protein disulfide isomerase (PDI) has a typical CXXC active site motif, which has been mimicked by linear (Moroder et al, 1996;, backbone-cyclized and photoactive peptides (Cattani-Scholz et al, 2002). These peptides form relatively strained rings when disulfide bonded, thereby increasing the reduction potential and facilitating thiol/disulfide exchange.…”

Methods for the Peptide Synthesis and Analysis

“…In comparative studies among several resins on the synthesis of human stromal cell-derived factor (SDF)-1α [24] and the acyl carrier protein (ACP [65][66][67][68][69][70][71][72][73][74] [25], higher purities were obtained with CM than with PS supports when using similar loadings. Compatibility of all these PEG-based resins with aqueous buffers allows their use for biochemical applications such as on-resin screening of chemical libraries and the development of affinity chromatography [26][27][28][29].…”

“…In small peptides bearing more than one disulfide bridge, regioselective strategies are usually sought [64,65], although usually various strategies needs to be tested to know the most favorable arrangement of Cys protecting groups. In bigger peptides and proteins, the kinetics of folding dominate the process and usually the concomitant oxidation of all Cys gives the correct folded molecule [66].…”