Fluorescence resonance energy transfer by S-layer coupled fluorescence dyes

Abstract

In this paper two fluorescence dyes were coupled to surface layer (S-layer) proteins of Lysinibacillus sphaericus A12 and Lysinibacillus sphaericus B53 to easily generate a fluorescence resonance energy transfer (FRET). S-layer proteins are structure proteins which self-assemble in aqueous solutions, on surfaces and at interfaces forming 2D-paracrystalline structures with a defined symmetry in nanometer range. These properties and the fact, that a lot of modifiable functional groups are available on their surface, make them a perfect coating and binding matrix for the generation of functionalized surfaces, e.g. needed for a sensor assembly. Here we chemically link two fluorescence dyes, which are able to perform a FRET, to S-layer proteins by carbodiimide-crosslinking chemistry. Fluorescence dyes were coupled to the protein with a yield of around 54mol%, demonstrating a modification of every second protein monomer if fluorescence dyes are statistical distributed. A FRET could be detected between the two fluorescence dyes when linked to protein polymers whereas no FRET could be detected if fluorescence dyes are linked to protein monomers. This demonstrates, that the polymer structure is essential for FRET and that fluorescence dyes are statisticaly distributed on protein polymers with a close proximity of donor and acceptor dye. Due to the fact that the used S-layer proteins build a unit cell of p4 symmetry, it can be assumed that two fluorescence dyes are linked to one unit cell. In this paper the FRET pair arrangement and its optimization is described in which the FRET efficiency can be increased from 6 to 40%, simply by varying the molar ratio of donor:acceptor. In result a sensory surface can be generated and used for detection of numerous substances in water like pharmaceuticals or heavy metals.