Photophysical characterization of interchromophoric interactions between rhodamine dyes conjugated to proteins

Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E. coli sliding clamp ( clamp) with emphasis on the properties of the H-dimers that form when the dimeric protein is labeled with one dye at each side of the dimer interface. Overall, results are consistent with an equilibrium between non-emissive dimers and unstacked monomers that experience efficient dynamic quenching Protein constructs labeled with tetramethylrhodamine show the characteristic features of H-dimers in their absorption spectra and a c.a. 40-fold quenching of fluorescence intensity. The degree of quenching decreases when samples are labeled with a tetramethylrhodamine derivative bearing a six-carbon linker. H-dimers do not form in samples labeled with Alexa 488 and A546, but fluorescence is still quenched in these samples through a dynamic mechanism. These results should help researchers design and interpret fluorescence experiments that take advantage of the properties of rhodamine dimers in protein research.