Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties
The rapid development of new microscopy techniques for cell biology has exposed the
need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the
same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent
lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright
Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component
of fluorescence lifetime (FL~0.66 ns) and exceptional fluorescence brightness, was designed
for FLIM experiments. Here, we present the X-ray structure and discuss the structure-functional
relations of BrUSLEE. Its development from the EGFP (enhanced green fluorescent proteins) precursor
(FL~2.83 ns) resulted in a change of the chromophore microenvironment due to a significant
alteration in the side chain conformations. To get further insight into molecular details explaining the
observed differences in the photophysical properties of these proteins, we studied their structural,
dynamic, and electric properties by all-atom molecular-dynamics simulations in an aqueous solution.
It has been shown that compared to BrUSLEE, the mobility of the chromophore in the EGFP is
noticeably limited by nonbonded interactions (mainly H-bonds) with the neighboring residues.