(405g) Directed Evolution of Bright Mutants of a Flavin Dependent Anaerobic Fluorescent Protein From Pseudomonas Putida

We report the construction of bright variants of an anaerobic fluorescent protein (AFP) reporter from Pseudomonas putida [1]. Fluorescent reporter proteins are widely used as non-invasive cellular probes to study gene expression, protein-protein interactions, subcellular localization, and protein trafficking. The green fluorescent protein (GFP) and its related analogs are routinely employed for in vivo imaging. However GFPs require molecular oxygen for fluorescence, thereby restricting their use to anaerobic niches. Recently, a novel class of flavin mononucleotide (FMN) dependent fluorescent proteins from Pseudomonas putida and Bacillus subtilis was reported [2]. The flavin-dependent fluorescent proteins employ a buried FMN chromophore for fluorescence in an oxygen-independent manner. Therefore FMN based fluorescent proteins are attractive candidates for live cell imaging in anaerobic conditions.

In this work, we describe the engineering of two spectrally improved mutants of the flavin-binding anaerobic fluorescent protein from Pseudomonas putida that show a two-fold increase in fluorescence emission relative to the parent protein. To isolate these bright mutants, we randomly mutagenized key amino acids by saturation mutagenesis with degenerate nucleotides and screened the mutant library by spectrofluorometric measurements for spectral shifts and improved brightness. The amino acids were selected for mutation based on their proximity (up to 0.3 nm) to the buried FMN chromophore in a molecular model of the three-dimensional protein structure constructed by homology modeling. We found that brightness enhanced mutants were both mutated at the same amino acid – a flavin proximal phenylalanine residue (F37), and we designated these mutants as AFP F37S and F37T. Aromatic amino acids are known to quench aromatic fluorophores through stacking interactions. We therefore hypothesize that the two-fold enhancement in fluorescence that results from the F->S and F->T mutations in the bright variants is a consequence of a decrease in quenching of flavin fluorescence by the aromatic phenylalanine residue in the wild type protein. We corroborate this claim by demonstrating that mutating the wild type F37 residue to the aromatic amino acids tryptophan (F37W) and tyrosine (F37Y) abrogates the native fluorescence significantly.

To the best of our knowledge, our study is the first report of engineering of a flavin-based anaerobic fluorescent protein for prokaryotic expression. Further engineering of anaerobic fluorescent proteins for spectral enhancements would enable studies of real time gene expression, protein localizations, and dynamics in obligate anaerobes as well as in hypoxic niches of the human body (e.g. hypoxic tumors), that have thus far been hindered by the strict requirement of O₂ for GFP to fluoresce. 

References:

1. A. Mukherjee, C.M. Schroeder, submitted to Biotechnology and Bioengineering (2011). 
2. T. Drepper, et al. Nature Biotechnology, 25: 443:445 (2007).