Structure and Engineering of the S-Layer Protein SbsB

The goal of this project is to elucidate the molecular structure of a bacterial S-layer protein. S-layers are crystalline assemblies of identical (glyco)-proteins and constitute the outermost component of the cell wall of many bacteria. S-layers can fulfil important functions as a structural scaffold or as a virulence factor promoting cell adhesion. S- layers are also of biotechnological interest for e.g. the nanopatterning of surfaces. Despite their importance, little is known about the molecular structure of S-layer proteins and no crystal structure of any S-layer protein is available to date. This project will explore the structure-function relationship of the S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2, a thermophilic Gram-positive bacterium. A previous study examined aspects of the molecular structure of SbsB and identified 23 residues, which are located at the surface of the SbsB monomer. The proposed project will expand this line of research by determining the position for each of the 23 residues. Specifically, residues will be identified which are located within the pores, on the inner cell wall-bound or outer side of the SbsB S-layer lattice, as well as at the subunit interface. Residues located at the subunit interface are of particular importance because these residues are involved in the assembly process. To identify residues located at each of the four surfaces, single cysteine mutants of the S-layer protein will be subjected to a differential surface accessibility screen using targeted chemical modification with sulfhydryl-reactive polymeric reagents. The screen will be performed on SbsB assembled into four substrates: Either monomeric, monomeric cell wall-bound, assembled or assembled cell wall-bound. Each of the four different substrates exposes a different set of surfaces to the ambient and this differential accessibility is the basis to determine the position of the residues. Another tool to pinpoint the position of residues will be the use of polymeric reagents of different size. For example, polymers with a hydrodynamic diameter higher than the pore lumen will predominantly have access to residues at the outer surface (or inner surface) of the S-layer lattice, while smaller polymeric reagents will also permeate into the pore. In summary, the project will yield detailed knowledge about the structure-function relationship of SbsB and this will constitute a significant advance over the previous studies on S-layer proteins. In addition, the project will provide mutants suitable to generate crystals for X-ray structure analysis. Finally, the project will produce engineered S-layer proteins with a photochemical switch to control their assembly.