Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Abstract

Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.

Insights into the mechanisms of light‐oxygen‐voltage domain color tuning from a set of high‐resolution X‐ray structures

“…Therefore, we conclude that irrespective of the buffer, the C-terminus of the Q148K variant is unlatched and Lys148 occupies the outermost position. These findings are corroborated by the structure of the Q489K variant of iLOV described in the accompanying article 56 .…”

“…Surprisingly, none of the mutations produced a red shift in the absorbance or fluorescence excitation spectra, and the observed blue shifts were smaller than those resulting from Gln→Leu replacement in other proteins 36 . Thus, significant color tuning would probably require two or more simultaneous mutations 45,56 .…”

“…As described in the accompanying article 56 , CagFbFP-Q148K was crystallized using the precipitant solution containing 0.1 M MES monohydrate pH 6.5 and 12% w/v PEG 20000.…”

“…POINTLESS and AIMLESS 58 were used to merge, scale and assess the quality of the data, as well as to convert intensities to structure factor amplitudes and to generate Free-R labels. As described in the accompanying article 56 , datasets 6YX6 and 6YXC revealed strong anisotropy and were processed using STARANISO 59 using the following criterion for determining the diffraction-limit surface: <Imean/σImean> of more than 2.…”

“…As described in the accompanying article 56 , we were able to obtain three types of Q148K variant crystals, all using the precipitant solutions with pH of 6.5. Unexpectedly, and contrary to the results of previous simulations 43,44 , in all of the obtained models the C-terminus of the protein is clearly unlatched, and the side chain of Lys148 is extended away from FMN (Figure 5A,B,C).…”