Crystal preparation of fully deuterated red fluorescence protein for neutron diffraction experiment

柴崎 千枝; 安達 基泰*

Shibazaki, Chie; Adachi, Motoyasu*

The green fluorescent protein derived from the jellyfish Aequorea victoria is well-known as a fluorescent protein. A red fluorescent protein absorbs orange light and emits red fluorescence. mRuby derived from the sea anemone Entacmaea quadricolor exhibits strong red emission, significant Stokes shift, short maturation time from chromophore precursor to fluorophore, and high stability as a monomer. Its excellent pH stability and photostability also make it a valuable protein for cellular imaging and for visualizing molecular interactions through FRET. Our aim is to investigate the ionization and protonation states of the dissociable groups of the chromophore and surrounding amino acid residues, and the orientation of water molecules using neutron diffraction experiments. Here, mRuby protein was expressed in E. coli cultured in heavy water in addition to light water. We conducted X-ray diffraction experiments after crystallization, obtaining diffraction data at 0.93A (pH 8.0) and 1.09A (pH 3.7) for hydrated protein, and diffraction data at 1.04A (pH 8.0) for deuterated protein. In this poster, we will report the differences in stability between hydrated and deuterated mRuby, as well as the results of the structural analysis.