Homo-dimeric spherulin 3a: A single-domain member of the beta gamma-crystallin superfamily

The beta gamma-crystallin superfamily of eye lens proteins comprises a clas s of structurally related members with a wide variety of different function s. Common features of these proteins are 1. the Greek-key motif of antipara llel beta-sheets, called the crystallin fold, and 2. the high intrinsic lon g-term stability, Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within th e beta gamma-crystallin family, Based on sequence homology and 'domain swap ping', it has been proposed to represent an evolutionary ancestor of presen t-day eye lens crystallins, Since S3a is highly expressed in spherulating p lasmodia of P. polycephalum under a variety of stress conditions, it can be assumed that the protein may serve as a compatible solute in the cytosol o f the slime mold, In order to investigate the stability and other physicoch emical properties of a single-domain all-beta protein, we isolated natural S3a, For the large-scale purification, the recombinant protein was cloned a nd expressed in Escherichia coli, The detailed spectral and biochemical ana lysis proved the recombinant protein to be authentic. In its native form, S 3a is dimeric, Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the form ation of higher oligomers, In order to preserve the native quaternary struc ture without aggregation artifacts in denaturation/renaturation experiments , the Cys4-->Ser mutant (S3a C4S) was produced, Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties. A t pH 3 - 4, both proteins form a stable compact intermediate (A-state). Con centration-dependent thermal and chemical denaturation showed that the equi librium unfolding of S3a obeys the simple two-state model with no significa nt occurrence of folding intermediates.