(594o) Characterization of Flavin-Binding Fluorescent Proteins As a New Class of Oxygen-Independent Biological Imaging Probes

In this work, we report comprehensive biochemical and biophysical characterization of a new class of oxygen-independent flavin-binding fluorescent proteins (FbFPs) to enable their robust application as versatile biological imaging probes. Genetically encodable fluorescent reporters have catalyzed significant advances in our understanding of biological processes. By enabling live cell imaging with exquisite spatiotemporal resolution, fluorescent reporter proteins can monitor in a minimally invasive fashion, intracellular protein expression, localization, trafficking, and protein-protein, and protein-nucleic acid interactions. However, existing fluorescent proteins are almost exclusively derived from the green fluorescent protein (GFP) family. GFPs require molecular oxygen for fluorescence, which excludes their application to investigating anaerobic bioprocesses. Recently, an alternative class of fluorescent imaging probes was developed that bind flavin mononucleotide (FMN) as their chromophore and are capable of fluorescence in an oxygen-independent manner [1,2]. Therefore, flavin-binding fluorescent proteins (FbFPs) are attractive candidates for live cell imaging in microaerobic and anaerobic conditions.

In previous work, we have engineered FbFPs by directed evolution to improve brightness of fluorescence emission [3]. Here, we cloned, expressed, and purified existing FbFPs identified in B.subtilis, P.putida, and A.thaliana and characterized their performance as fluorescent reporters with respect to several key parameters. Specifically, we determined quantum yield, oligomeric state, fraction of fluorescent holoprotein in solution, intracellular stability, and denaturation/maturation kinetics. In addition, we evaluated sensitivity of fluorescence in FbFPs to environmental conditions such as temperature, pH, and redox. Furthermore, we investigated their potential as transcriptional reporters by employing FbFP-tagged promoters to monitor gene expression in E.coli cells under different conditions of growth. Based on our results, we identified iLOV, (from A.thaliana), as the most suitable FbFP for biological imaging applications. Overall, our characterization data brings to light several properties of FbFPs that are highly desirable in fluorescent reporter proteins, e.g., thermal tolerance, broad pH range, redox insensitivity, small size, and fast maturation. Combined with oxygen-independent fluorescence, FbFPs represent a new and powerful class of fluorescent reporter proteins with potential applications in cancer therapy, bioremediation, and metabolic engineering for the production of high value biomolecules like bio-derived fuels and polyhydroxyalkanoates (PHA). We expect our work  to enable the widespread use of FbFPs as reporters as well as provide a biochemical and biophysical basis for further protein engineering to optimize their spectral properties.

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, K.B. Weyant, and C.M. Schroeder, submitted.