Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability

Abstract

Background: iLOV is a fluorescent flavoprotein engineered from the plant blue light receptor phototropin. Results: Structures reveal altered protein-chromophore interactions within the flavin-binding cavity of iLOV when compared with its progenitors. Directed evolution further anchored the chromophore to increase iLOV photostability by an order of magnitude. Conclusion: Improving iLOV photostability by constraining its fluorophore establishes a framework for fine-tuning fluorescence. Significance: Enhanced photostability increases iLOV utility as an oxygen-independent fluorescent reporter.

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…Since the first development of LOV-based FPs 8,9 , several improved variants have been generated 10,12 . In this work, we thus comparatively analyzed the photophysical properties of BsFbFP and EcFbFP, both engineered from Bacillus subtilis YtvA, as well as PpFbFP, a derivative of Pseudomonas putida PpSB2-LOV, as bacterial LOV-based FPs.…”

“…PpFbFP variants Y112L and Q116V were constructed by saturation mutagenesis (details are described in the materials and methods section), and in addition, the plant-LOV based FPs, miniSOG and phiLOV2.1 were further characterized. These fluorescent reporter proteins were derived from the phototropin2 LOV2 domain of A. thaliana and were shown to either efficiently generate singlet oxygen upon blue-light irradiation 12 or exhibit improved photostability 10 .…”

“…Therefore, LOVbased FPs could successfully be established within research fields where the size of the fluorescent label is critical. For example, the use of iLOV as alternative fluorescent tag enabled advanced studies of the localization, movement and dynamics of target proteins and viruses 9,10,31,32 . Furthermore, in vivo and in vitro studies demonstrated that, in contrast to GFP-like proteins, LOV-based reporter proteins rapidly gain their fluorescence-active conformation because of their fast folding kinetics and the spontaneous incorporation of the chromophore 14,33 , thereby enabling their application as real-time reporters.…”

“…Their rapid assembly as well as the robustness of the fluorescence signal have rendered LOV-based FPs valuable quantitative reporters for biotechnological approaches [33][34][35][36][37] . To further establish LOV-based FPs as alternative fluorescent proteins, some of their biochemical and photophysical properties, including the fluorescence quantum yield, thermal stability and photobleaching susceptibility have been optimized in different studies by directed evolution approaches and sitedirected mutagenesis 9,10,12,38,39 and were subsequently determined with individual techniques. However, quantitative conclusions from spectrometry, cytometry and microscopy data vitally depend on the accurate characterization of their spectral properties that have been obtained under defined and comparable conditions.…”

“…Here the most prominent example is the incomplete autocatalytic synthesis of the chromophore in the absence of molecular oxygen, leading to the accumulation of inactive, non-fluorescing reporter proteins 6,7 . To conquer this limitation, alternative fluorescent proteins that are based on blue-light photoreceptors of the LOV (light oxygen voltage) family, have been developed recently [8][9][10][11] . These cyan-green fluorescing proteins bind flavin mononucleotide (FMN) as the chromophore and are either derived from bacterial photoreceptors (FMN-binding fluorescent proteins; FbFP 8 ) or are derivatives of the Arabidopsis thaliana phototropin 2 LOV2 domain (iLOV, miniSOG; 9,12 ).…”