We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATPdependent interaction of the motor protein myosin II (the crossbridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly (Ϸ300 s) and precisely (Ϸ0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step (Ϸ3.4 -5.2 nm) occurs immediately after myosin binding and is followed by a smaller step (Ϸ1.0 -1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family.isoforms ͉ optical tweezers ͉ single molecule I t has been know for a long time that the rate of cross-bridge cycling, defining shortening velocity (V), is fundamental for the biological role (1, 2) and the energy consumption of skeletal muscle (3). The shortening velocity of skeletal muscle varies very widely in relation to the isoforms of myosin II (1, 2), conferring on muscles the remarkable capability to adapt their performance to the requirements of posture, movement, and locomotion, by a modulation of isoforms gene expression (1). Shortening velocity and, in turn, the rate of ATP splitting (4) by skeletal myosins strictly depend on the load applied (Fenn effect) (5). The load sensitivity is likely to play a significant role in coordinating the action of the multiple individual motors that in skeletal muscle work cooperatively in a complex ensemble.It is widely accepted that, during its lifetime of attachment to actin (t on ), myosin goes through a conformational change of amplitude ␦ (working stroke) coupled to the release of the products of ATP hydrolysis [inorganic phosphate (Pi) and ADP]. Actin filaments, therefore, slide at an average velocity V ϭ ␦͞t on (6, 7). The rate of ADP release (k ϪD ) has been suggested to define both the isoform dependence (8, 9) and the load sensitivity (10) of shortening velocity through a modulation of t on . However, no definitive evidence of such role has been provided for skeletal...

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers' spatial and temporal resolution down to today's values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 μs of actin binding by β-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.