Events & Seminars > Event Details


4:00 pm
Room 304, Chemistry Building

Fluorescence Polarization and Fluctuation Analysis (FPFA): A method for simultaneously measuring fluorescence lifetime, time-resolved anisotropy, fluorophore concentration, molecular brightness, and lateral diffusion times


Dr. Steve Vogel
National Institutes of Health

Hosted by: Professor Jim Demas

Both Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) can be used to study protein interactions and protein-complex conformational changes in living cells. FRET can detect the proximity between two fluorophores on a 1-10 nm scale. It can be measured by monitoring a change in the fluorescence lifetime of a FRET donor for Hetero-FRET, or by monitoring fluorescence anisotropy for Homo-FRET between two or more identical fluorophores. FCS can measure the concentration of a fluorophore in solution, molecular brightness (the number of fluorophores associated with a complex in solution), and the correlation time (an attribute sensitive to the mass and shape of a protein complex). We have developed a hybrid single-molecule method that simultaneously measures fluorescence lifetime (for Hetero-FRET measurements), time-resolved anisotropy (for Homo-FRET and rotational diffusion time measurements), and FCS (for measuring fluorophore concentration, molecular brightness, and lateral diffusion time measurements. This new form of spectroscopy is named Fluorescence Polarization and Fluctuation Analysis (FPFA). I will review how FPFA can be used to: 1) monitor protein-protein interactions, 2) detect conformational changes in a protein complex, 3) screen for mutations affecting protein complex assembly, and 4) monitor two independent FRET reactions simultaneously.