24] Fluorescence resonance energy transfer imaging microscopy and fluorescence polarization imaging microscopy

Abstract

Publisher Summary This chapter describes the fluorescence resonance energy transfer imaging microscopy and fluorescence polarization imaging microscopy. Measurements of fluorescence resonance energy transfer (FRET) efficiency between a donor–acceptor pair can provide unique information about changes in molecular proximity and structural dynamics within a protein complex. A particularly attractive feature of the FRET approach is that these proximity measurements can be conducted within large, functional molecular complexes under physiological conditions. In particular, the chapter describes how FPIM studies of single molecular complexes may be used to experimentally determine the value of the orientation factor (κ 2 ). In principle, κ 2 values can also be measured by FPIM for other protein systems by covalently cross-linking the donor- and acceptor-labeled proteins to a unique site on actin, for example, through Cys-374. The knowledge of the κ 2 value greatly improves the precision of FRET-based distance determinations and it may be used as a new parameter for studying protein structural dynamics.