Biochemical study of fluorescence resonance energy transfer using purified hybrid fluorescent proteins

초록

Fluorescence resonance energy transfer (FRET) is a physical phenomenon in which energy is transferred between two fluorophores in close proximity to each other. FRET occurs when the excited state energy of a donor fluorophore is transferred to an acceptor fluorophore, which subsequently emits light at a longer wavelength than the donor. FRET is a widely used technique in biochemistry to study the interactions between biomolecules, such as proteins or nucleic acids, in living cells or in vitro. FRET can be used to measure the distance between two fluorophores, which is typically in the range of 1 to 10 nanometers. Changes in the distance and conformation of the molecules in response to various stimuli, such as ligand binding, enzymatic activity, or protein-protein interactions can be monitored by placing the fluorophores on specific positions of the biomolecules of interest. We have studied FRET characteristics of various fusion proteins of fluorescent proteins in vitro with purified hybrid fluorescent proteins. Bacterial expression vectors for hybrid fluorescent proteins were generated using combinations of Blue/Green/Red fluorescent proteins. Hybrid fluorescent proteins were purified using nickel-NTA column affinity chromatography.