(818c) Engineering Biological Photoreceptors As Fluorescent Molecular Probes for Bioimaging

We report on the development of naturally occurring biological photoreceptors as an advanced class of fluorescent imaging probes with broad applications in synthetic biology, biological engineering, biomedical, and industrial bioprocess platforms. The elegant photophysical properties of natural photoreceptors were recently harnessed to develop genetically encoded imaging probes known as flavin-based fluorescent proteins (FbFPs) [1,2]. FbFPs are characterized by two key advantages relative to the widely used green fluorescent protein (GFP): namely, small size (≈ 50 % of GFP) and oxygen-independent maturation of fluorescence [1,2]. However, FbFPs are at a nascent stage of development and a complete understanding of their performance and properties as a practical set of biological probes is lacking. Importantly, FbFPs have modest brightness, which further limits pervasive applications of FbFPs for biological imaging.

With a view to advancing FbFP-based imaging technology, we systematically addressed the aforementioned issues. First, we extensively characterized the existing set of FbFPs in terms of key biophysical parameters, such as brightness (quantum yield), oligomeric state, maturation time, fraction of (fluorescent) holoprotein, pH tolerance, redox sensitivity, and thermal stability. We demonstrated that FbFPs show important advantages as broad-spectrum imaging probes, including broad pH tolerance (pH 4-11) and thermal stability (up to 60˚C) as well as rapid maturation of fluorescence (≈ 3 min. vs. ≈ 30 min. in case of GFP) [3]. Second, we applied protein engineering using directed evolution to develop bright mutants of FbFPs. Specifically, we constructed and screened a library of approximately 30,000 FbFP variants and isolated two FbFP mutants (F37S and F37T) that displayed a 2-fold increase in overall brightness of fluorescence emission [4]. Our work demonstrated that enhancements in FbFP probe brightness can be engineered through improvements in quantum yield as well as increasing the strength of association between the protein and its fluorescent flavin cofactor. In addition, we demonstrated that aromatic amino acids in the fluorophore (flavin) binding pocket of FbFPs can potentially quench probe fluorescence. Finally, we validated the application of FbFPs as stable and noninvasive fluorescent reporters of gene expression in vivo by constructing a series of FbFP-based transcriptional fusions to probe promoter dynamics in Escherichia coli under different conditions of growth [3].

Overall, our results identify FbFPs as an emerging class of fluorescent protein probes that are characterized by stable cellular expression and robust performance under a wide range of environmental conditions, such as lack of oxygen and extremes of pH and temperature. Importantly, we develop a molecular engineering framework to identify and engineer new and improved FbFP-variants using directed evolution based on site saturation mutagenesis. In summary, we anticipate that our work will encourage the continued development of FbFPs and enable their broad application as fluorescent reporters of gene expression, protein localization, and whole cell microbial sensors in conditions where existing imaging probes fail to perform optimally (e.g., fast time-scale cell processes, extremophilic microbes, anaerobic bioprocesses such as fermentation).

References

1. T. Drepper, et al. Nature Biotechnology, 25: 443:445 (2007).

2. S. Chapman, et al. PNAS, 105(50):20038:20043 (2008)

3. A. Mukherjee, et al. PLOS ONE, accepted (2013)

4. A. Mukherjee, et al. Journal of Biological Engineering, 6(1):20 (2012)