Fluorescent S-Layer Fusionproteins; Reassembling Behaviour and Spectral Properties

Abstract

S-layer proteins are crystalline bacterial cell surface layer proteins. When they are isolated or genetically modified with functional moieties they are still able to reassemble spontaneously into their distinctive monomolecular lattice symmetry as originally found on the cell surface. In solution they form differently sized self-assembly products while they build crystalline nanostructured biocoatings o nsolid supports. In our case we fused the pH-dependent green fluorescent protein (EGFP) in a 1:1 stoichiometry with a truncated form of the S-layer protein SgsE from Geobacillus stearothermophilus NRS2004/3a. We investigated the formation of the 2D nanostructured monomolecular crystalline self-assembly products with the functional moiety in defined position and orientation, representing a new patterning material for biomolecular construction kits. With transmission electron microscopy (TEM) we could demonstrate that isolated SgsE-EGFP subunits build mono- and double-layered self-assembly products, forming differently sized flat sheets and cylinders with p2 lattice symmetry (a=11.9+/-0.6nm, b=14.7+/-0.6nm, g=81.2+/-1.1nm). Atomic force microscopy (AFM) was used to resolve the crystalline domain nanostructure of the fluorescent S-layer proteins on functionalised solid supports. We demonstrated the application as building blocks by coating silica particles with the fluorescent S-layer protein, fabricating in this way a pH-dependent nanostructured surface-coating. The pKa value of the pH-dependent fluorescent S-layer coating was calculated after measuring the EGFP fluorescence emission in different pH environements using flow cytometry and fluorescence microscopy. The colloidal behaviour was investigated with Zeta-potential measurements. In conclusion, these fluorescent S-layer fusion protein assemblies can be used for investigating systematically changes in pH and surface potential at the nanoscale.