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

Abstract

Muscle contraction can be explained by the swinging lever-arm model. However, the dynamic features of how the myosin head swings the lever-arm and its initial interactions with actin are not well understood even though they are essential for the muscle force generation, contraction speed, heat production, and response to mechanical perturbations. This is because myosin heads during force generation have not been directly visualized. Here, we engineered thick filaments composed of DNA origami and recombinant human muscle myosin, and directly visualized the heads during force generation using nanometer-precision single-molecule imaging.We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet.Upon strong binding, the head two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. These results can explain all mechanical characteristics of muscle contraction and suggest that our DNA origami-based assay system can be used to dissect the mechanistic details of motor proteins.

The synergic role of actomyosin architecture and biased detachment in muscle energetics: insights in cross bridge mechanism beyond the lever-arm swing

“…Next, we explore how much preferential detachment and attachment separately influence the ratio when the geometrical hindrance is included. Both properties have been introduced theoretically in [6] and, more recently, gained experimental support [21,22,24,31]. We first note that eliminating the preferential attachment (Figure 3, lower panel, circles) does not affect significantly the ratio, but maintains values even higher than in the previous analysis.…”

“…We tested the model predictions directly by observing the in vitro effect of biased kinetics when the acto-myosin spacing is preserved. To achieve our goal, we used the DNA-origami technique [24,35], a nanotechnology suitable to construct arbitrary three-dimensional nanostructures from DNA. Ten DNA duplexes were bundled in parallel [24] to build a rod nanostructure (DNA rod) to which we attached myosin motors with precise spacing of 14.3 nm (Figure 4A).…”

“…In the model these are described by the attachment and detachment rates between the detached active state d ON and each of the attached states a x (Figure 1A). At least two experimentally verified components influence the apparent attachment and detachment rates at different ν : the preferential detachment of compressed myosin motors [21,22] and the existence of a limited binding region on the double-helix conformation of the actin filament, as suggested by single molecule experiments [23] and more recently by fast AFM images [24]. The latter component generates geometrical hindrance to the actomyosin interaction due to the spatial mismatch between myosin motors and actin filament periodicities (Figure 1B and C).…”

Crystalline chitin hydrolase is a burnt-bridge Brownian motor

“…Complex functions of life are the results of chemical and physical reactions supported by enzymes [ 1 ]. Motor proteins such as kinesin [ 2 ], dynein [ 3 ], and myosin [ 4 , 5 ] transport a substance or generate a force by linear movement along with the rail proteins. In other cases, F o F 1 -ATP synthase generates ATP from a proton motive force [ 6 ] and V o V 1 -ATPase pumps ions [ 7 , 8 ] by rotating motions in a membrane.…”

Molecular goniometers for single-particle cryo-EM of DNA-binding proteins

“…In 2006, Rothemund introduced DNA origami, a powerful approach to building megadalton-sized nanostructures that relies on a long single-strand DNA (ssDNA) 'scaffold' to template the assembly of numerous short DNA oligonucleotide 'staples' into a custom shape 8 . This method has since been used to build several innovative custom instruments helpful for measuring and identifying forces in biological systems [9][10][11][12] . As a way to address an important challenge of cryo-EM-difficulty in aligning small particle images-we aimed to correctly determine each 2D particle orientation by prescribing the orientation using a nanoscale device: a molecular goniometer made out of DNA origami.…”