Ruprecht-Karls-Universitšt Heidelberg

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Single molecules

Figure 1: Schematic experimental set-up: actin filaments move over a surface of myosin or heavy meromyosin
Figure 2 Movement of fluorescently labeled actin filaments recorded with a highly sensitive CCD camera

Molecular dynamics in the in vitro motility assay

The ability of heart and skeletal muscle to contract is based on the fundamental interaction of the two contractile proteins actin and myosin. This basic interaction can be studied in the in vitro motility assay originally devised by Kron and Spudich (1986), where fluorescently labeled actin filaments move over a surface of immobilised myosin or heavy meromyosin. The motion of actin filaments is recorded with a highly sensitive fluorescence imaging set-up.

Many parameters of this motion have been shown to be of significant importance for our understanding of the acto-myosin interaction, as e.g. the filament velocity is thought to be directly correlated to the unloaded shortening velocity of muscle fibers and therefore a direct reflection of the cross-bridge turnover rate. Also this assay is ideally suited to screen the functional domains of myosin, such as the nucleotide binding site, the actin binding site, the converter region and the lever arm. Furthermore myosin isoforms and actin mutants can be selectively studied in this assay.

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Force measurements with optical tweezers

Optical tweezers is the name of a technique of holding small polystyrene beads in the focus of a laser beam. The diffraction of the laser at the bead surface produces a force that always directs the bead towards the laser focus. This effect is simply due to the geometry of the configuration and can be applied to measure very small forces, like the force between a single myosin and actin molecule.