Microsecond conformational dynamics of biopolymers studied by two-dimensional fluorescence lifetime correlation spectroscopy
Many biological functions of biopolymers (protein/DNA/RNA) are realized with their spontaneous structural fluctuation. Therefore, the elucidation of the energy landscape of biopolymers and their structural dynamics is essential. Single molecule spectroscopy is a powerful tool to investigate this problem, and the single molecule Förster resonance energy transfer (smFRET) technique is widely utilized. However, conventional smFRET detects only millisecond and slower dynamics. In this presentation, I’ll introduce recently developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)1,2 that distinguishes the conformers by their fluorescence lifetime and detects their interconverstion dynamics with a microsecond time resolution. Next, I will introduce a new method that combines dynamic fluorescence quenching (DQ) and 2D FLCS. In comparison to FRET which detects relatively large structural change, DQ is more sensitive to the local solvent accessibility of the attached dye. A major advantage of this method is that it requires only single-dye labeling compared to FRET which requires double-labeling. By applying this DQ 2D FLCS to a singly-labeled DNA hairpin, we succesfully resolved the open and closed forms in the equilibrium and detected their microsecond interconversion dynamics. I’ll also show the application of 2D FLCS on preQ1 riboswitch, an important antibiotic drug target, to resolve its heterogeneous folding dynamics and distinct ligand binding mechanisms.
[1, 2] Ishii, K. & Tahara, T. Journal of Physical Chemistry B, 2013 117, 11414 & 11423.