The molecular structure of green fluorescent protein

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Title: The molecular structure of green fluorescent protein
Author: Yang, Fan
Advisor: Phillips, George N., Jr.
Degree: Doctor of Philosophy thesis
Abstract: The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods using selenomethionyl GFP crystals. The protein is in the shape of a cylinder, comprising 11 strands of $\beta$-sheet with an $\alpha$-helix inside and short helical segments on the ends of the cylinder. This motif, with $\beta$-structure on the outside and $\alpha$-helix on the inside, represents a new protein fold. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with oxidation of its C$\sb{\alpha}$-C$\sb{\beta}$ bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests which side chains could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein. GFP can be reduced by sodium dithionite and as a result, loses its fluorescence. The structure of reduced GFP has been solved which shows that the side chain of Tyr66 at the fluorophore is the group being reduced. While oxidized, the C$\sb{\alpha}$ atom of Tyr66 is not a chiral center since all fluorophore atoms are co-planar. After reduction, the C$\sb{\alpha}$ atom returns to a chiral center, but has either an L or D configuration, indicating that the breakage of the resonance system eliminates the fluorescence of GFP and the reduction of Tyr66 is not stereospecific.
Citation: Yang, Fan. (1997) "The molecular structure of green fluorescent protein." Doctoral Thesis, Rice University.
Date: 1997

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