YPet through the eyes of mCLIFY.

Fluorescent proteins (FP) have become widely used biophysical and cell biological tools that report on a variety of processes within cells. The popular yellow FP, YPet, serves as either a donor or acceptor for many FRET-based biosensors, yet it has been difficult to fully characterize. In studying our circularly permuted and equally bright variant, mCLIFY, features common to YPet were revealed, including β-barrel and chromophore sequence homology. We determined the atomic structure of mCLIFY to 1.6 Å resolution, which reveals a remarkably well-aligned π-interaction between the chromophore with Tyr37, as well as a water-mediated network of hydrogen bonds which supports the chromophore. Both features are likely promoting the robust photophysical properties of mCLIFY. To better understand the self-association properties of YPet, known to be a weak dimer, sedimentation velocity analytical ultracentrifugation (SV-AUC) determined a monomer-dimer dissociation constant KD of 3.4 μM, whereas mCLIFY did not dimerize. Size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering and multi-angle light scattering (SEC-SAXS-MALS) confirmed these findings. SAXS studies show that mCLIFY correlates well to the solution monomer, whereas YPET exists as an antiparallel dimer in solution. Their fluorescence intensities and anisotropies were compared via FLIM (Fluorescence Lifetime Imaging Microscopy) of FPs expressed in E. coli. Protein concentrations increased above 5 μM for both FPs within 45 minutes of IPTG induction. Unexpectedly, average anisotropy of YPet significantly decreased from 0.322 to 0.183 with increasing protein concentration presumably due to HOMO-FRET and/or trivial reabsorption of emitted photons. Anisotropy for mCLIFY (0.316) decreased at much higher concentrations. The data presented here suggest that potential quantitative errors from overexpression of non-monomeric FPs may be avoided with newly available, more fully studied monomeric variants like mCLIFY.