Stability and Self-Assembly of the S-Layer Protein of the Cell Wall ofBacillus stearothermophilm

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Abstract
The surface layer of the cell envelope of B. stearothermophilus consists of a regular array of protein subunits. As shown by sodium dodecyl sulfate polyacrylamide gel-electrophoresis and ultracentrifugation, the fully solubilized S-layer protein represents a homogeneous entity with a subunit MW of 115 .+-. 5 kilodalton. Solubilization of the protein may be accomplished at acid pH, or using high concentrations of urea or guanidine .cntdot. HCl. It is accompanied by (partial) denaturation, thus interfering with the characterization of the protein in its unperturbed native state. Removal of the solubulizing agent by dialysis or dilution allows the the S-layer to be reassembled into 2-dimensional crystalline lattices identical to those observed in intact cells. To determine the kinetics of association, optimum conditions are found to be rapid mixing with 0.1 M sodium phosphate pH 7.0, 20.degree. C, final protein concentration > 10 .mu.g/ml. If the time course of the self-assembly is monitored by light scattering, and by chemical cross-linking with glutardialdehyde, multiphasic kinetics with a rapid initial phase and slow consecutive processes of higher than 2nd-order are observed. The rapid phase may be attributed to the formation of oligomeric precursors (MW > 106). Concentration-dependent light scattering measurements give evidence for a critical concentration of association, suggesting that patches of 12-16 protein subunits fuse and recrystallize into the final (native) S-layer structure. Recrystallization tends to be complete.