Professor Achillefs Kapanidis

Abstract:

We present a single-molecule method for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) of single diffusing molecules. An application of this method to the study of a bacterial transcription initiation complex is presented.

Abstract:

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.