Revealing Myosin's Power Stroke with High-Speed Scattering Interferometry

Abstract

How nature addresses brownian motion and achieves high processivity of molecular motors is a fundamental question in biophysics. Single molecule techniques are particularly suited to investigate nanoscopic motion with high spatial accuracy, but have struggled to provide information about structural dynamics associated with the individual motor steps. Fundamentally, the challenge has been to achieve both nanometer spatial accuracy and microsecond temporal resolution without considerably interfering with the mechanics of the step. Here, we show that interferometric scattering microscopy (iSCAT) can track the motion of both the head and stalk of myosin 5a in vitro without need of a drastic reduction of ATP concentration. We achieved 1 nm spatial precision and sub-millisecond temporal resolution using quantum dot-sized metallic nano-particles as labels. In this way, we could visualize conformational changes during the myosin 5 processive movement, a waiting state and brownian search when encountering obstacles - fundamental components of the stepping mechanism of myosin 5a. Remarkably, we observe tight control of lever-arm diffusion and motion for the duration of the step. Moreover, iSCAT signal brings additional information about z-position of the label which completes the 3D picture of myosin's moment. Our results illustrate how iSCAT can be used as a new approach to study dynamic structural changes of biomolecules in action.