Print Email Facebook Twitter The S-layer protein of Lactobacillus acidophilus ATCC 4356: Identification and characterisation of domains responsible for S-protein assembly and cell wall binding Title The S-layer protein of Lactobacillus acidophilus ATCC 4356: Identification and characterisation of domains responsible for S-protein assembly and cell wall binding Author Smit, E. Oling, F. Demel, R. Martinez, B. Pouwels, P.H. Centraal Instituut voor Voedingsonderzoek TNO Publication year 2001 Abstract Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (SA-protein) of 43 kDa. SA-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a = 118 Å; b = 53 Å, and γ = 102°) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of SA-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of SA-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of SA-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic SA-protein. A calculated SA-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei. © 2001 Academic Press. Chemicals/CAS: 1,2-dioleoylphosphatidylserine, 70614-14-1; Bacterial Proteins; Membrane Glycoproteins; Membrane Proteins; Peptide Fragments; Phosphatidylserines; Recombinant Fusion Proteins; Solutions; surface array protein, bacteria; Trypsin, EC 220.127.116.11 Subject BiologyAmino Acid SequenceBacterial ProteinsCell WallCrystallizationElectrophoresis, Polyacrylamide GelEscherichia coliLactobacillus acidophilusMembrane GlycoproteinsMembrane ProteinsMicroscopy, ElectronModels, MolecularMolecular Sequence DataPeptide FragmentsPhosphatidylserinesProtein BindingProtein Structure, QuaternaryProtein Structure, SecondaryProtein Structure, TertiaryRecombinant Fusion ProteinsSequence AlignmentSequence Analysis, ProteinSolutionsTrypsinEscherichia coliLactobacillus acidophilusLactobacillus caseiLactobacillus crispatusLactobacillus helveticus To reference this document use: http://resolver.tudelft.nl/uuid:84d8c382-7e26-4867-a041-361d09f3a803 DOI https://doi.org/10.1006/jmbi.2000.4258 TNO identifier 41810 ISSN 0022-2836 Source Journal of Molecular Biology, 305 (2), 245-257 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.