Biophysical characterization of the entire bacterial surface layer protein SbsB and its two distinct functional domains.

The crystalline bacterial cell surface layer (S-layer) protein SbsB of Geobacillus stearothermophilus PV72/p2 was dissected into an N-terminal part defined by the three consecutive S-layer homologous motifs and the remaining large C-terminal part. Both parts of the mature protein were produced as separate recombinant proteins (rSbsB(1-178) and rSbsB(177-889)) and compared with the full-length form rSbsB(1-889) (rSbsB). Evidence for functional and structural integrity of the two truncated forms was provided by optical spectroscopic methods and electron microscopy. In particular, binding of the secondary cell wall polymer revealed a high affinity dissociation constant of 3 nm and could be assigned solely to the soluble rSbsB(1-178), whereas rSbsB(177-889) self-assembled into the same lattice as the full-length protein. Furthermore, thermal as well as guanidinium hydrochloride induced equilibrium unfolding profiles monitored by intrinsic fluorescence, and circular dichroism spectroscopy allowed characterization of rSbsB(1-178) as an alpha-helical protein with a single cooperative unfolding transition yielding a DeltaG value of 26.5 kJ mol(-1). The C-terminal rSbsB(177-889) could be characterized as a beta-sheet protein with typical multidomain unfolding, which is partially less stable as stand-alone protein. In general, the truncated forms showed identical properties compared with the full-length rSbsB with respect to structure and function. Consequently, rSbsB is characterized by its two functionally and structurally separated parts, the specific secondary cell wall polymer binding rSbsB(1-178) and the larger rSbsB(177-889) responsible for formation of the crystalline array.