The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active‐site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.

Inhibitors of urokinase-type plasminogen activator (uPA) were selected in vitro from two ecotin phage-display libraries to study the effect on binding of amino acid substitutions at critical positions 108, 110, 112, and 113 within the 100s loop (RNKL, respectively, in wild type ecotin). The first, a focused library, was the result of a computation-assisted approach using the three-dimensional structure of the ecotin-trypsin complex to guide the modeling of amino acid substitutions predicted to increase affinity for uPA. The second, a complete library, allowed for all substitutions at the above identified positions. The consensus sequences selected from the focused, and complete libraries were RRWS and R(R/N)QL, respectively. Inhibition constant determinations showed ecotin variants containing these sequences to be similarly potent (K i ‫؍‬ 1-2 nM). These substitutions were combined with previously identified substitutions in another critical region of ecotin. One of these combinations (D70R/M84R/RRQL) is the tightest (K i ‫؍‬ 50 pM) ecotin variant inhibitor of uPA. The blending of combinatorial methods and computer algorithms designed to predict stronger binders has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. This technology can be applied to select for enhanced binding interactions at protein-protein interfaces and accelerate the process of protease inhibitor development.The formation of protein-protein complexes is an essential element of virtually every cellular process. These complexes may be permanent or transient, associating and dissociating as conditions require. Enzyme-protein inhibitor complexes and activator complexes are archetypal examples of transient complexes (1, 2). The biochemical study of numerous enzymeinhibitor complexes has led to the assembly of structural and mechanistic data bases, providing further impetus for using these complexes as models to investigate fundamental biochemical and biophysical principles of protein-protein recognition.The interaction of urokinase-type plasminogen activator (uPA) 1 with the macromolecular serine protease inhibitor, ecotin, is the basis for an enzyme-inhibitor complex model system that is particularly amenable to genetic manipulation. uPA is a serine protease originally found to convert plasminogen to plasmin (3), thereby playing an active role in extracellular proteolysis, cell migration, and tissue remodeling (4). As a result of its potential role in cancer metastasis and tumor invasion (5), uPA has become an important target for anti-cancer drug development (6).Ecotin is a macromolecular serine protease inhibitor found in the periplasm of Escherichia coli (7). This highly malleable, dimeric, macromolecular serine protease inhibitor has been modified to enhance inhibition of selected proteases (8 -11). Ecotin has four surface loops that participate in tetramer formation through their interaction with two bound protease molecules. The interaction involves two binding sites on each ecoti...

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