Biophysical Characterization of the Entire Bacterial Surface Layer Protein SbsB and Its Two Distinct Functional Domains

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 ) 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 ␣-helical protein with a single cooperative unfolding transition yielding a ⌬G value of 26.5 kJ mol ؊1 . The C-terminal rSbsB 177-889 could be characterized as a ␤-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.Crystalline bacterial cell surface layers (S-layers), 1 representing the outermost cell envelope component of many bacteria and archaea, are composed of a single species of identical protein or glycoprotein subunits forming a lattice with either oblique, square, or hexagonal symmetry (1-3). S-layer subunits can be isolated from cell wall fragments by disintegration with high concentrations of hydrogen bond-breaking agents (e.g. GdmHCl). The intrinsic ability of isolated S-layer subunits to recrystallize into their original regular arrays either in suspension or on surfaces (e.g. noble metals, lipid films (4)) makes S-layer proteins unique building blocks in molecular nanobiotechnology and biomimetics (5-9). The mature S-layer protein SbsB (10) of Geobacillus stearothermophilus PV72/p2 (11) is processed from a 920-amino acid-long preprotein by cleavage of a 31-residue-long signal peptide and assembles into an oblique (p1) lattice type. Recently, SbsB has been used to generate chimeric S-layer proteins by fusion of SbsB with streptavidin either on the N-terminal or on the C-terminal end of SbsB (12). These chimeric S-layer proteins combine both the ability to form monomolecular protein lattices on different surfaces and the binding of biotinylated compounds. Such a functional biomolecular matrix with repetitive features in the subnanometer range allows a unique approach ...

The Surface Location of Individual Residues in a Bacterial S-Layer Protein

“…7,[18][19][20] The self-assembly process of monomeric S-layer proteins to two-dimensional protein crystals has been studied using chemical cross-linking 21 and AFM. 10 The proteins' ability to self-assemble is usually localized within one or more crystallization domains, which can be dissected from the cell-wall anchoring domain, as shown by genetic or proteolytic truncation experiments 1,[22][23][24][25][26] and supported by insertion mutagenesis studies. 26,27 Relatively little is known about the molecular nature of the interactions between S-layer subunits.…”

“…Two of the pores are round and small with an internal diameter of 1 nm and two are larger and oval shaped with dimensions of 3.5 nm × 1.5 nm. The propensity for assembly resides in the C-terminal 712-aminoacid-long crystallization domain of SbsB, 22 while the N-terminal anchoring domain (residues 32 to 208) binds to a secondary cell wall polymer (SCWP) that is covalently linked to the peptidoglycan sacculus within the cell wall. 32 In the previous study, a chemical approach was used to investigate the structure of SbsB.…”

Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein

“…SbsB is expected to consist of fibronectin type III and Ig-like domains, structures of known mechanical stability, which allows for the assumption that the double peak originates from the unfolding of such domains in the protein. Thermally and chemically induced unfolding studies also indicated that SbsB is a multidomain protein exhibiting a complex unfolding behavior, with a three-step unfolding having been observed (16). A contour length increase of about 12 nm indicates that the consecutive unfolding intermediates are closely located in the structure of the protein.…”

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

“…This specific molecular interaction is often mediated by a recurring structural motif of approximately 55 amino acids, which is mostly found in triplicate at the N-terminus of S-layer proteins. These so-called SLH motifs are involved in cell wall anchoring of S-layer proteins by recognizing a distinct type of SCWP which carries pyruvic acid residues [32,112,[136][137][138][139][140]. Moreover, the coexistence of two N-terminally located binding domains for SCWPs and peptidoglycans was also described for the SLH domain carrying S-layer protein of G. stearothermophilus PV72/p2 (SbsB) [29,141].…”