ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

Abstract

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.single ATP turnover assay | ATP dwell times | dwell time distribution | TIRF microscopy F orces and movements, generated when myosins interact with actin filaments, are driven by the coupling of conformational changes in the myosin head domain to particular steps of the ATP hydrolysis cycle (1-3). Essential steps of the ATP hydrolysis cycle for skeletal muscle myosin and acto-myosin were characterized in solution studies (1,4,5). With the concept of Hill (6), it became possible to relate solution studies to mechanical, biochemical, and structural studies on muscle fibers (7, 8) to form a general concept for the coupling of ATP hydrolysis to the generation of mechanical work (6, 9).In studies on large ensembles of myosin molecules such as solution and fiber studies, however, crucial reaction steps could be masked by the ensemble averaging and thus may complicate relating solution kinetics to structural changes and the generation of forces and movements. In addition, ensemble studies can be complicated by mixed populations of myosin molecules-for example, by the presence of different myosin isoforms or different posttranslational modifications. Here we explored the feasibility to study ATPase kinetics from ...

Kinetic mechanism ofNicotiana tabacummyosin‐11 defines a new type of a processive motor

“…This suggested that the high duty ratio of Nt 175kDa myosin-11 may result from a rate-limiting ADP release step of the actin bound state of the myosin. Our results from the in vitro motility experiments with low and high [Mg 2+ ] free concentrations indicate the existence of Mg 2+ -sensitive states that influence ADP release to a similar extent as reported for processive myosin-5 motors (31,67,68).…”

Structural Oscillations of Non–muscle Myosin II–C2: Time Resolved Confocal Microscopy

“…Similar kinds of time periods have been observed recently by Amrute‐Nayak et al. for individual myosin molecules due to ATP turnover at the active site . They found two types of dwell times for the myosin molecules‐one shorter time of ∼0.5 sec and a longer time ∼3‐4 sec.…”

Direct visualization of human myosin II force generation using DNA origami-based thick filaments

“…A correction of background noise was performed by calculating the average intensity of the perimeter around the ROI 53 . We measured the on-time by fitting the data to a double exponential decay and defined the slower rate as the ATP binding rate, thus giving a complete ATPase cycle 43 . The ATP binding rate of Cy3-EDA-ATP was corrected to the rate of normal ATP by a factor of 2.8 42 .…”