Mapping the molecular structure of the S-layer protein SbsB.
Doctoral thesis, UCL (University College London).
Surface layer (S-layer) proteins form 2-dimensional crystalline structures at the cell surface of most eubacteria and all archaea. S-layers are generally composed of a single self-assembling (glyco)protein species and can fulfil a number of roles including shape maintenance, exo-enzyme adhesion, and virulence promotion. Engineered S-layers carrying functional inserts at high density also show potential in nanotechnology. However, there is currently no complete structure at atomic resolution available for any S-layer protein. This work is engaged in the (i) structural elucidation of the SbsB S-layer protein of Geobacillus stearothermophilus by determining the surface location of individual residues. A cross-linking screen was used to analyse 23 cysteine residues, known to be surface accessible in the monomer, with respect to their position within the assembled S-layer. The study was able to distinguish between eight residues that are positioned close to the subunit-subunit interface and 10 residues located at the cell-wall facing inner surface. The assay also confirmed the previous assignment of four residues to the outer ambient-exposed surface. The tolerance of the individual sites to insertion of a short peptide sequence was investigated to (ii) create chimeric S-layer proteins for nanobiotechnological applications and (iii) advance the structural elucidation of SbsB. Insertion mutagenesis at nine of the surface sites led to mutants of conserved tertiary structure, of which six were assembly-positive. The six proteins provide model constructs for the incorporation of functional tags into S-layer arrays for nanobiotechnology application as high-density vaccine carriers. The mutagenesis screen also revealed three mutants of conserved tertiary structure but of assembly-negative phenotype. The potential of forming 3D crystals for X-ray crystallography was tested with one mutant yielding small crystals. In conclusion, my work has advanced the understanding of the molecular structure of S-layer proteins and provided the basis for future structural investigations.
|Title:||Mapping the molecular structure of the S-layer protein SbsB|
|UCL classification:||UCL > School of BEAMS > Faculty of Maths and Physical Sciences > Chemistry|
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