Proteins display a diverse range of functions, from self-assembly to catalysis, owing to their ability to adopt unique structures through a wide variety of natural and unnatural amino acid building blocks. Labelling proteins is essential in these applications, as it allows for precise tracking of their behaviour, interactions, and functionality within complex systems. Fluorescent labelling of biomolecules remains one of the widely adopted techniques. Traditional methods of fluorescent tagging proteins involve chemical labelling or recombinant expression alongside a fluorescent protein. These small molecule fluorophores are typically employed in excess which further necessitates additional downstream processing steps including separation of the conjugated molecules with the unreacted fluorophores. On the other hand, recombinantly appended fluorescent proteins to the biomolecule of interest, likely affects the functioning of the labelled protein due to its large size. There is still a need to develop a recombinant technique to label proteins with smaller molecular weight fluorophores which are genetically encodable.
To overcome this, we utilized PhoCl1, a green-to-red photoconvertible fluorescent protein, as a recombinant fusion protein tag. Upon exposure to 405 nm light, PhoCl1 undergoes cleavage, producing an N-terminal empty barrel and a nine-amino-acid C-terminal peptide fragment (CTPF) containing the chromophore. We demonstrate that the CTPF aggregates, resulting in a red fluorescence emission. We leverage this property of PhoCl1, where the CTPF, now bound to the biomolecule of interest, results in the protein of interest acquiring fluorescence after photocleavage. We successfully demonstrated this approach of conferring fluorescence to a silica binding peptide (R5) and a riboflavin kinase enzyme (MjRibK), both of which exhibited red fluorescence upon photocleavage. The fluorescence spectra, quantum yields, lifetime of the red fluorescent peptide/protein was determined. MALDI and SDS-PAGE was performed to determine the purity of the red fluorescent biomolecule. Finally, we showed that this tagging method does not affect the structural and functional activity of the peptide or protein.
We anticipate that this approach of inducing red fluorescence in non-fluorescent biomolecules will open new possibilities for labelling and studying biomolecules in various applications.
