Although it is accepted that the vertebrate two-domain betagamma-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.
The ␥-crystallin superfamily of eye lens proteins comprises a class of structurally related members with a wide variety of different functions. Common features of these proteins are 1. the Greek-key motif of antiparallel -sheets, called the crystallin fold, and 2. the high intrinsic long-term stability. Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within the ␥-crystallin family. Based on sequence homology and 'domain swapping', it has been proposed to represent an evolutionary ancestor of present-day eye lens crystallins. Since S3a is highly expressed in spherulating plasmodia 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 of the slime mold. In order to investigate the stability and other physicochemical properties of a single-domain all- protein, we isolated natural S3a. For the large-scale purification, the recombinant protein was cloned and expressed in Escherichia coli. The detailed spectral and biochemical analysis proved the recombinant protein to be authentic. In its native form, S3a is dimeric. Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the formation of higher oligomers. In order to preserve the native quaternary structure without aggregation artifacts in denaturation/renaturation experiments, the Cys4 ǞSer mutant (S3a C4S) was produced. Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties. At pH 3 -4, both proteins form a stable compact intermediate (A-state). Concentration-dependent thermal and chemical denaturation showed that the equilibrium unfolding of S3a obeys the simple two-state model with no significant occurrence of folding intermediates. Key words: ␥-Crystallin superfamily / Greek-key motif / Long-term stability / Physarum polycephalum / Two-state model.Proteins of the ␥-crystallin superfamily are closely related in their sequence as well as in their gene and protein structure, all consisting of domains with two Greek-key motifs of antiparallel -sheets. They show high long-term stability, in the case of the eye lens crystallins without turnover or degradation over the whole life time of the organism (Jaenicke, 1994). Based on sequence homology, the encystment-specific spherulin 3a (S3a) from the slime mold Physarum polycephalum was predicted to be structurally related to the N-terminal domain of ␥B-crystallin (Bernier et al., 1987;Wistow, 1990). Although the sequence identity of the two proteins is no more than 20%, all amino acid residues essential for the ␥-crystallin fold are found to be conserved. The predicted structural similarity was confirmed by 3D NMR (Rosinke et al., 1997).Being the only known member of the ␥-crystallin family with only one domain, S3a is a particularly suitable molecule for biophysical studies on the anomalous stability of the all- crystallin fold observed f...
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