title: Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies.
abstract: Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (<or=2.4 A), and possibly low-barrier, hydrogen bond between the chromophore hydroxyl and introduced Asp148.
authors.affiliation: Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403-1229, USA.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Shu
authors.forename: Karen
authors.initials: K
authors.lastname: Kallio
authors.forename: Xinghua
authors.initials: X
authors.lastname: Shi
authors.forename: Paul
authors.initials: P
authors.lastname: Abbyad
authors.forename: Pakorn
authors.initials: P
authors.lastname: Kanchanawong
authors.forename: William
authors.initials: W
authors.lastname: Childs
authors.forename: Steven G
authors.initials: SG
authors.lastname: Boxer
authors.forename: S James
authors.initials: SJ
authors.lastname: Remington
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D049452:Green Fluorescent Proteins