Introduction

Many prokaryotic organisms have regular arrays of (glyco)proteins on their outermost surface (for a compilation, see ref. 1). These monomolecular crystalline surface layers, termed S-layers (2), are found in members of nearly every taxonomic group of walled bacteria and cyanobacteria and represent an almost universal feature of archaeal cell envelopes (see Fig. 1). S-layers are generally composed of a single protein or glycoprotein species with a molecular mass of

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press inc., Totowa, NJ

Fig. 1. Electron micrographs of freeze-etching preparations of whole cells of (A) B. sphaericus, showing a square S-layer lattice; (B) Thermoplasma thermo-hydrosulfuricus, revealing a hexagonally ordered array. Bars: (A) 200 nm; (B) 100 nm. (Reprinted from ref. 45 with permission from the publisher; © 2001, Elsevier Science.)

Fig. 1. Electron micrographs of freeze-etching preparations of whole cells of (A) B. sphaericus, showing a square S-layer lattice; (B) Thermoplasma thermo-hydrosulfuricus, revealing a hexagonally ordered array. Bars: (A) 200 nm; (B) 100 nm. (Reprinted from ref. 45 with permission from the publisher; © 2001, Elsevier Science.)

40,000 to 230,000 Daltons and exhibit either oblique (p1, p2), square (p4), or hexagonal (p3, p6) lattice symmetry with unit cell dimensions in the range of 3 to 30 nm (see Fig. 2). One morphological unit consists of one, two, three, four, or six identical subunits, respectively. The monomolecular arrays are generally 5 to 10 nm thick and show pores of identical size (diameter of 1.5-8 nm) and morphology. In most S-layers, the outer face is less corrugated than the inner face. Moreover, S-layers are highly anisotropic structures regarding the net charge and hydrophobicity of the inner and outer surface (3,4). Owing to the crystalline character of S-layers, functional groups (e.g., carboxyl, amino, hydroxyl groups) are repeated with the periodicity of the protein lattice.

Fig. 2. Schematic representation of types of S-layer lattice grouped according to possible 2D space group symmetries. Morphological units were chosen arbitrarily and are shown in dark gray. (Reprinted from ref. 8 with permission from the publisher; © 2003, Wiley-VCH.)

Because S-layers possess a high degree of structural regularity and the constituent subunits are the most abundant of all bacterial cellular proteins, these crystalline arrays are excellent models for studying the dynamic aspects of assembly of a supramolecular structure in vivo and in vitro. Moreover, the use of S-layers provided innovative approaches for the assembly of supramolecu-lar structures and devices. S-layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological and chemically synthesized molecules or nanoparticles. In this context, one of the most important properties of isolated S-layer (glyco)protein subunits is their capability to reassemble into monomolecular arrays in suspension, at the air interface, on a solid surface, on floating lipid monolayers (see Fig. 3), and on liposomes or particles (for a review, see refs. 5-8).

An important line of development in S-layer-based technologies is presently directed toward the genetic manipulation of S-layer proteins. These strategies open new possibilities for the specific tuning of their structure and function. S-layer proteins incorporating specific functional domains of other proteins while maintaining the self-assembly capability will lead to new ultrafiltration

Fig. 3. (A) Schematic illustration of recrystallization of isolated S-layer subunits into crystalline arrays. The self-assembly process can occur (B) in suspension, (C) at the air-liquid interface, (D) on solid supports, and (E) on Langmuir lipid films. (Reprinted from ref. 5 with permission from the publisher; © 1999, Wiley-VCH.)

Fig. 3. (A) Schematic illustration of recrystallization of isolated S-layer subunits into crystalline arrays. The self-assembly process can occur (B) in suspension, (C) at the air-liquid interface, (D) on solid supports, and (E) on Langmuir lipid films. (Reprinted from ref. 5 with permission from the publisher; © 1999, Wiley-VCH.)

membranes, affinity structures, enzyme membranes, metal-precipitating matrices, microcarriers, biosensors, diagnostics, biocompatible surfaces, and vaccines (6,7).

Although up to now most S-layer technologies developed concerned life sciences, an important emerging field of future applications relates to nonlife sciences. Native or genetically modified S-layers recrystallized on solid supports can be used as patterning elements for accurate spatial positions of nanometer-scale metal particles or as matrices for chemical deposition of metals as required for molecular electronics and nonlinear optics.

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