Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution

Abstract

Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

“…The gain in contrast is limited by stray light entering the objective at subcritical angles. Further improvements could be achieved with the more favourable beam characteristics from a laser [10] and dichroic mirrors that reflect a maximal proportion of the incident light [4]. However, compared with the prism-based method [4,15], TIRF via the objective lens allows an optimal objective-to-specimen working distance as well as free access to the opposite surface for the photolysis beam.…”

“…Finally, we would like to point out that, although single molecule detection has been reported and opens up new possibilities with regard to mechanochemical coupling studies of motor proteins [4], the major discrepancies in the literature concern sliding over assemblies of myosin heads at low loads [6,7]. Thus, it may be desirable to follow the kinetics of nucleotide turnover by a few hundred adjacent myosin heads during unloaded sliding.…”

“…To this end, recent eflbrt has been directed towards in vitro motility assays where mechanical events have now been detected at the level of single molecular interactions between actin and myosin [1][2][3]. Likewise, the sensitivity of ATPase assays have been improved so that the long-lived myosin product complex (M.ADEP~) can be detected at the single molecule level [4]. The problem remains, however, of determining the kinetics of the ATPase at this level of sensitivity under conditions of actin activation.…”

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

“…These include e.g., a modified version of flow cytometry [63,64 ] used for single fluorophore detection and nearfield microscopy [65][66][67], evanescent-field fluorescence microscopy (EFFM, also called total internal reflection microscopy [68]) [69], and versions of epi-fluorescence microscopy [70][71][72][73] used for single fluorophore imaging. In the case of low temperature experiments, reviewed in Ref.…”

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

“…Single‐molecule fluorescence measurement is especially effective for investigating the dynamic reaction and motion of biomolecules 6, 7, 8, 9, 10. One of the most successful approaches is the observation of fluorescence resonance energy transfer between a single pair of fluorophores (smFRET) 11, 12, 13, 14.…”