Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies

Wild type green fluorescent protein (wt-GFP) and the variant S65T/HI48D 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 (similar to 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/HI48D, the A band absorbance maximum is red-shifted to similar to 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 (similar to 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/HI48D, 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 (<= 2.4 angstrom), and possibly low-barrier, hydrogen bond between the chromophore hydroxyl and introduced Asp148.