Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus.
Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies.
Kinns
Helen
H
Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK.
Badelt-Lichtblau
Helga
H
Egelseer
Eva Maria
EM
Sleytr
Uwe B
UB
Howorka
Stefan
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D011994:Recombinant Proteins; C104635:SbsB protein, Bacillus stearothermophilus