No abstract

An astonishingly broad application potential in biotechnology, biomimetics, and nanotechnology is revealed by studies on the structure, chemistry, biosynthesis, genetics, self-assembly, and function of supramolecular surface layers (S layers). These are monomolecular, crystalline assemblies of protein or glycoprotein subunits and represent one of the most commonly observed surface structures of prokaryotic cell envelopes (see schematic representation of an archaebacterial cell envelope).

Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding D-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component of many bacteria and archaea (35,37,38). S-layers are composed of identical protein or glycoprotein subunits, and they completely cover the cell surface during all stages of bacterial growth and division. The S-layer subunits assemble into either oblique, square, or hexagonal lattices. In the case of Bacillaceae, the N-terminal part is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer (SCWP) to the rigid cell wall layer (2,5,21,25,34,35).S-layers are unique biomaterials with properties most relevant for applications in molecular nanotechnology, nanobiotechnology, and biomimetics (38). Many S-layer proteins recrystallize into regularly structured monolayers on solid supports, such as silicon wafers, gold chips, and silanized glass or plastic materials, as well as on Langmuir lipid films, on liposomes (20,23), and at the air-water interface. Pores passing through S-layer lattices are of identical size and morphology, and functional groups show a regular distribution and high density. For production of S-layer-based biosensors (38), affinity microparticles (45), and solid-phase immunoassays (3,4,39), functional groups in the S-layer lattice were exploited as covalent binding sites for biologically active macromolecules, such as enzymes, antibodies, or ligands. As alternatives to the existing technology, namely, immobilization by chemical methods, genetic approaches are particularly attractive for incorporation of functional peptide sequences into S-layer proteins, which must be done at positions that do not interfere with their self-assembly properties and their interaction with SCWP.

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archeaobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.