4.7 Article

Identifying Assembly-Inhibiting and Assembly-Tolerant Sites in the SbsB S-Layer Protein from Geobacillus stearothermophilus

期刊

JOURNAL OF MOLECULAR BIOLOGY
卷 395, 期 4, 页码 742-753

出版社

ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.10.012

关键词

bacterial exoprotein; supramolecular assemblies; S-layer; insertion mutagenesis; electron microscopy

资金

  1. Biotechnology and Biological Sciences Research Council
  2. Nuffield Foundation
  3. Department of Chemistry at University College London
  4. University College London
  5. Austrian Science Foundation [P18510-B12]
  6. European Union [NASSAP 13523]
  7. Austrian Science Fund (FWF) [P18510] Funding Source: Austrian Science Fund (FWF)
  8. Biotechnology and Biological Sciences Research Council [BB/E010466/1] Funding Source: researchfish
  9. BBSRC [BB/E010466/1] Funding Source: UKRI

向作者/读者索取更多资源

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 win 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. (C) 2009 Elsevier Ltd. All rights reserved.

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