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Journal of Bacteriology, May 2009, p. 3339-3349, Vol. 191, No. 10
0021-9193/09/$08.00+0     doi:10.1128/JB.01782-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer{triangledown}

Heikki Vilen,1 Ulla Hynönen,1 Helga Badelt-Lichtblau,2 Nicola Ilk,2 Pentti Jääskeläinen,3 Mika Torkkeli,4 and Airi Palva1*

Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, P.O. Box 66, FIN-00014 University of Helsinki, Helsinki, Finland,1 Center for NanoBiotechnology, BOKU University of Natural Resources and Applied Life Sciences Vienna, A-1180 Vienna, Austria,2 Centre of Excellence in Computational Complex Systems Research, Department of Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology (HUT), P.O. Box 9203, FIN-02015 HUT, Espoo, Finland,3 Department of Physics, Division of Materials Physics, P.O. Box 64, FIN-00014, University of Helsinki, Helsinki, Finland4

Received 19 December 2008/ Accepted 11 March 2009

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many Eubacteria and most Archaea species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium Lactobacillus brevis ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG)12 (molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW = 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of Lactobacillus S-layers.

* Corresponding author. Mailing address: Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, P.O. Box 66, FIN-00014 University of Helsinki, Helsinki, Finland. Phone: 358 9 191 57058. Fax: 358 9 191 57033. E-mail: Airi.Palva{at}Helsinki.fi

{triangledown} Published ahead of print on 20 March 2009.

Journal of Bacteriology, May 2009, p. 3339-3349, Vol. 191, No. 10
0021-9193/09/$08.00+0     doi:10.1128/JB.01782-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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