Decay time shortening of fluorescence from donor–acceptor pair proteins using ultrafast time-resolved fluorescence resonance energy transfer spectroscopy

Abstract

We improved an ultrafast time-resolved fluorescence resonance energy transfer (FRET) spectroscopy system and measured directly the decrease in the fluorescence decay time of the FRET signal, without any entanglement of components in the picosecond time scale from the donor–acceptor protein pairs (such as cameleon protein for calcium ion indicator, and ligand-activated GRIN–Go proteins pair). The drastic decrease in lifetime of the donor protein fluorescence under the FRET condition (e.g. a 47.8% decrease for a GRIN–Go protein pair) proves the deformation dynamics between donor and acceptor fluorescent proteins in an activated state of a mixed donor–acceptor protein pair. This study is the first clear evidence of physical contact of the GRIN–Go proteins pair using time-resolved FRET system. G protein-coupled receptors (GPCRs) are the most important protein family for the recognition of many chemical substances at the cell surface. They are the targets of many drugs. Simultaneously, we were able to observe the time-resolved spectra of luminous proteins at the initial stage under the FRET condition, within 10 ns from excitation. This new FRET system allows us to trace the dynamics of the interaction between proteins at the ligand-induced activated state, molecular structure change and combination or dissociation. It will be a key technology for the development of protein chip technology.