Crystal Structure Determination Of Two New Red Fluorescent Proteins By X-Ray Crystallography

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2017-08

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University of Hawaii at Manoa

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Recently scientists are interested in developing new variants of red fluorescent proteins (RFPs), because biosensors basing on RFPs show visual superiority in live-cell imaging. In this thesis, the structures of two RFP variants, K78BoF and mRuby0.4-3, were investigated by X-ray crystallography. A monomeric crystal structure of K78BoF was obtained at pH 6 with resolution at 2.13Å, while a dimeric crystal structure of mRuby0.4-3 was determined at pH 8.2 with resolution at 2.63Å. Both of their chromophores are formed by Met-Tyr-Gly with K78BoF in the cis state and mRuby0.4-3 in trans. Hydrogen bonds connecting water molecules and residues to K78BoF chromophore probably stabilize the conformation of the chromophore and contribute to the red fluorescence emitted. However, the substitution of an unnatural amino acid pboronophenylalanine breaks two hydrogen bonds, causing a deviation of the conformation. Analysis of hydrogen bonds on mRuby0.4-3 chromophore was limited by the relatively low resolution of the crystal.

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Red fluorescent protein, Crystallization

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