In the dimeric glutathione reductase (GR) from Escherichia coli,, the interface domain is largely surrounded by the other three domains in each subunit of the protein. Subgenes encoding three forms of the interface domain have been expressed in E. coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains. The isolated INT domain was found to be a soluble and folded protein, but it was isolated as a mixture of the dimer and at least two species of higher molecular weight. The latter were believed to arise by further association of the dimer via the newly exposed and unsatisfied hydrophobic contact regions. In the variant INTN, three hydrophobic residues normally involved in the contact with the NADPH-binding domain in GR were replaced. This partly suppressed the further aggregation of the dimers. However, continued aggregation at high protein concentrations suggested that at least one further site of unwanted aggregation was still present. After four additional amino acid replacements in the region normally in contact with the FAD-binding domain, the resulting variant INTFN exhibited no unspecific aggregation, even at concentrations as high as 3.2 mg/mL. The conformational stability of INTN and INTFN was not affected by these exchanges, as judged by superimposable cooperative GuCl-induced equilibrium unfolding transitions at 1.0 M GuCl. Conditions were also found for GR and for INTFN under which they dissociate into compact monomers. The effects on dimerization of mutations introduced at the subunit interface can therefore now be studied in both proteins. An equilibrium dissociation constant of 25 mu M was estimated for INTFN. This work shows that it is possible to generate a soluble, folded form of a normally buried protein domain by the rational introduction of a small number of amino acid exchanges at its newly exposed hydrophobic surfaces, without interfering with its intrinsic structure and stability. The repertoire of protein folds accessible to design and redesign is thus substantially increased.
