New Monomeric Bright Yellow Genetically Encoded Fluorescent Protein

Although, currently available fluorescent proteins (FPs) provide an exceptional variety of high-performance options, new variations with improved properties are emerging. YPet is considered to be the brightest of the YFP variants, but it has the tendency to form dimers or oligomers under physiological conditions, compromising quantification of biophysical signals such as anisotropy and FRET. Here, we introduce mCLFY, a new monomeric bright yellow FP based on circular permutation of the amino acid sequence of YPet, separating the N- and C-termini of mCLFY from the protective barrel caps to potentially improve chromophore stability. The new N- and C-termini of mCLFY are located on the opposite end of the β barrel at YPet residues 175 in the loop arising from β-strand 8 and β-strand 9 residue 176. Fluorescence correlation spectroscopy (FCS) yielded a diffusion constant for mCLFY of D = 111 ± 1 μm2/s (s.e.m., n = 14), whereas for YPet, D = 57 ± 2 μm2/s (n = 14). Native gel electrophoresis supports these observations showing faster migration of mCLFY compared to YPet. Quantum yield (QY) of mCLFY is 0.76, using the reported 0.77 QY of YPet as a standard. Molar extinction coefficients were determined at λ = 517 nm, ε = 121 ± 7 x 103 and 124 x 103 M−1·cm−1 for mCLFY and YPet, respectively. mCLFY shows reduced pH sensitivity of brightness relative to YPet. Structural details that lead to the improved stability of mCLFY require further investigation. Using time correlated single photon counting, we determined fluorescence lifetimes of mCLFY and YPet to be 3.29 ± 0.04 ns and 3.27 ± 0.09 ns (n = 8 ea.), respectively. mCLFY is an improved FP similar to YPet, but with less tendency to dimerize and less pH sensitivity.