Red Fluorescent Dyes and Proteins

For biomedical applications, red or near-infrared light is preferred because tissue is transparent to these wavelengths. However, traditional red fluorescent proteins exhibit weak emission, limiting their utility for in vivo applications. To overcome this limitation, we incorporate bright red-emitting cofactors into a stable protein scaffold, such as the heme nitric oxide/oxygen binding (H-NOX) protein from the thermophilic bacterium Caldanaerobacter subterraneus (Cs) or the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). Initial experiments have utilized phosphorus and silicon corrole complexes as the fluorescent cofactor. We are also exploring boron dipyrrins (BODIPY), which are popular fluorophores for biomedical applications due to their high fluorescence quantum yields and relatively narrow emission profiles. Since the BODIPY core itself emits green light, we are utilizing various functionalization strategies (such as halogenation, dimerization, and extension of the pi system) to red-shift the optical properties of BODIPY.

These synthetic fluorophores can then be reconstituted into the apo form of the H-NOX or HasA proteins. Alternatively, a special strain of E. coli, known as RP523, can be used to incorporate the fluorophore during protein expression. In order to investigate the protein­–cofactor interactions, the Lemon lab utilizes a variety of biophysical techniques in conjunction with site-directed mutagenesis to identify protein residues involved in fluorophore recognition and binding. Additionally, protein labeling experiments exploiting Förster resonance energy transfer (FRET) can provide information about the protein conformation. These complementary approaches enable us to develop a model for the protein­–cofactor interactions, guiding future experiments to enhance the properties of these protein conjugates.