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 proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern. This lack of welldefined substrate specificities makes the design of peptidebased highly selective inhibitors extremely difficult. However, the x-ray structure of the proteasome from Saccharomyces cerevisiae reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Structure-derived active site separation distances were exploited for the design of homo-and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a f lexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber. Spacer lengths were selected that satisfy the inter-and intra-ring distances for occupation of the active sites from the S subsites. X-ray analysis of the proteasome͞bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups. Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding. The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes. The present study confirms its utility also for inhibition of multicatalytic protease complexes.The proteasome is a multicatalytic protease complex that is involved in intracellular protein turnover in all three kingdoms of life. The proteasome is located in both the cytosol and the nucleus and acts in the degradation of abnormal, misfolded, or improperly assembled proteins, in stress response, cell cycle control, cell differentiation, metabolic adaptation, and cellular immune response. It also is involved in many pathophysiological processes like inflammation and cancer and constitutes a promising target for drug design. In mammals the proteasomes also are responsible for the production of the bulk of antigenic peptides, which are presented via MHC class I molecules on the cell surface to cytotoxic T lymphocytes. The antiviral cytokine INF-␥ induces transcription of three additional ␤ subunits (LMP2, MECL-1, and LMP7), which can replace their constitutive homologs (␤1, ␤2, and ␤5) in newly assembled proteasomes. The resulting immuno-proteasomes show altered cleavage patterns in vitro; these are thought to be essential for the proteasomal antigen processing (1). Most of these functions are linked to an ubiquitin-and ATP-dependent protein degradation pathway involving the 26S proteasome whose core and proteolytic chamber is formed by the 20S proteasome (2-7). The eukaryotic 20S proteasome consists of seven different ␣-type and seven different ␤-type subunits, all of which have been cloned and sequenced and can be grouped by sequence homology (8)....

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4-aminomethyl)phenylazobenzoic acid (H-AMPB-OH) and (4-amino)phenylazobenzoic acid (H-APB-OH) were synthesized and used to cyclize a bis-cysteinyl-octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H-AMPB-OH proceeded smoothly on a chlorotrityl-resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H-APB-OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S-tert-butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S-trityl protection. Side-chain cyclization of the APB-peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side-chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly.

Spot the difference: Conformational analysis of the 2S,4R and 2S,4S epimers of N‐acetyl‐4‐mercaptopyrrolidine‐2‐carboxylic acid methyl esters reveals ring‐pucker preferences that are opposite of those of the hydroxyproline derivatives (see scheme). Replacement of proline or hydroxyproline in polypeptides with the chalcogen analogue should allow for fine‐tuning of the complex interplay of noncovalent interactions, steric hindrance, and stereoelectronic effects.

Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.