Abstract
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.
Highlights
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
For both rSbsB and rSbsB1–178, an increase in anisotropy could be observed resulting from complex formation, which was in contrast to the S-layer homologous (SLH)-truncated rSbsB177–889, which did not bind at all
The anisotropy profiles for rSbsB and rSbsB1–178 upon binding to F-secondary (accessory) cell wall polymer (SCWP) were characterized by an initial plateau for the unbound fluorescein labeled SCWP (F-SCWP) followed by an anisotropy increase over a range of approximately 2 orders of magnitude in concentration, which is typical of binding in a 1:1 stoichiometry
Summary
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 (rSbsB1–178 and rSbsB177– 889) and compared with the full-length form rSbsB1–889 (rSbsB). SbsB possesses three N-terminal SLH motifs that recognize a secondary (accessory) cell wall polymer (SCWP) in the rigid cell wall as specific binding partner [21, 22] This glycan is mainly composed of N-acetylglucosamine and N-acetylmannosamine in a molar ratio of 2:1 and contains pyruvic acid residues [22]
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