Structure and mechanism of the photoactivatable green fluorescent protein.
Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.
Henderson
J Nathan
JN
Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403-1229, USA.
Gepshtein
Rinat
R
Heenan
Josef R
JR
Kallio
Karen
K
Huppert
Dan
D
Remington
S James
SJ
D000042:Absorption; D004583:Electrons; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D008027:Light; D008956:Models, Chemical; D008968:Molecular Conformation; D009154:Mutation; D010777:Photochemistry / Q000379:methods*; D011522:Protons; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D011522:Protons; D049452:Green Fluorescent Proteins