Abstract
BackgroundFluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes.ResultsIn this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence.ConclusionsTo increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.
Highlights
Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution
We isolated two mutants involving an flavin mononucleotide (FMN)-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness of fluorescence emission relative to the wild type protein
Homology modeling and design of site saturation mutagenesis flavin-binding fluorescent protein (FbFP) fluorescence is mediated by a buried FMN chromophore and the fluorescence emission of FMN is known to be sensitive to its microenvironment [41]
Summary
Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. The available palette of GFP-based fluorescent proteins is limited by a dependence on molecular oxygen, which mediates oxidation of a cyclic tripeptide chromophore that is strictly required for fluorescence [15,16]. In this way, GFP-family reporters require oxygen for fluorescence and do not fluoresce in anaerobic environments [17,18,19,20]. Flavin-binding photosensors have been implicated in mediating diverse functions in prokaryotes, including regulation of stress response and virulence and mediating cell adhesion [27,28]
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