Characterization and Structural Basis for the Brightness of mCLIFY: A Novel Monomeric and Circularly Permuted Bright Yellow Fluorescent Protein

We present mCLIFY: a monomeric, bright, yellow, and long-lived fluorescent protein (FP) created by circular permutation of YPet, the brightest yellow FP from Aequorea Victoria for use in cellular and in vitro single molecule studies. mCLIFY retains the enhanced photophysical properties of YPET as a monomer at concentrations ≤ 40 μM. In contrast, we determined that YPet has a dimerization dissociation constant ( K _D ^1-2 ) of 3.4 μM. Dimerization of YPet can cause homo-FRET, which underlies quantitative errors due to dimerization and homo-FRET. We determined the atomic structure of mCLIFY at 1.57 Å resolution and used its similarity with Venus for guided chromophore-targeted substitution studies to provide insights into its enhanced photophysical properties. The mutation V58L within the chromophore pocket improved quantum yield and extinction coefficient, making mCLIFY ~30% brighter than Venus. The extensive characterization of the photophysical and structural properties of YPet and mCLIFY presented here allowed us to reveal the basis of their long lifetimes and enhanced brightness and the basis of YPet’s dimerization.