title: Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein.
abstract: The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.
authors.affiliation: Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA. howorka@medicine.tamu.edu
authors.forename: S
authors.initials: S
authors.lastname: Howorka
authors.forename: M
authors.initials: M
authors.lastname: Sára
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Kuen
authors.forename: U B
authors.initials: UB
authors.lastname: Sleytr
authors.forename: W
authors.initials: W
authors.lastname: Lubitz
authors.forename: H
authors.initials: H
authors.lastname: Bayley
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002850:Chromatography, Gel; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins