In single molecule Frster resonance energy transfer (FRET) spectroscopy, kinetics and dynamics of molecular processes are typically determined by analyzing energy transfer efficiency calculated using fluorescence intensity from donor and acceptor dyes. To understand complex molecular interactions and conformational changes during binding and oligomerization processes, it is important to utilize all available information from single molecule measurements. Since FRET efficiency is related to the lifetimes of dyes, additional information of the process can be extracted by analyzing fluorescence intensity and lifetime together. However, for a fast process where individual states are not well separated in a trajectory, it is not simple to obtain lifetime information. In collaboration with Dr. Irina V. Gopich, we developed analysis methods that utilize both fluorescence intensity and lifetime information. 2D FRET efficiency-lifetime histogram method visualizes the correlation between the FRET efficiency and mean fluorescence delay times (fluorescence lifetimes), which reveals the presence of the sub-microsecond dynamics in each state and the microsecond to millisecond kinetics between states. A more quantitative analysis method is the maximum likelihood method that determines the FRET efficiencies and the kinetics and lifetime parameters accurately. We have utilized the 2D FRET efficiency-lifetime analysis method to probe conformations of oligomers of the tetramerization domain (TD) of the tumor suppressor protein p53. TD is known to exist as a monomer at a low concentration and form a dimer and a tetramer sequentially as the concentration is raised. Our focus in this project is the characterization of the monomer and dimer for which atomic-resolution structures are not available. We have found that the tetramer dissociation constant is so low that individual oligomers cannot be clearly separated. Using single-molecule spectroscopy, it is possible to detect individual oligomers in equilibrium. To probe conformational changes and interactions between monomers simultaneously, we have developed three-color FRET. Combination of two- and three-color FRET experiments and the fluorescence lifetime analysis allows for a quantitative comparison of the oligomer conformations. The monomer is intrinsically disordered as expected and the dimer conformation is very similar to that of the tetramer but the C-terminus of the dimer is less structured and more flexible.
| Chung, Hoi Sung (2018) Transition Path Times Measured by Single-Molecule Spectroscopy. J Mol Biol 430:409-423 |
| Chung, Hoi Sung; Meng, Fanjie; Kim, Jae-Yeol et al. (2017) Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET. Proc Natl Acad Sci U S A 114:E6812-E6821 |
| Chung, Hoi Sung; Louis, John M; Gopich, Irina V (2016) Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins. J Phys Chem B 120:680-99 |
