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.