ABSTRACT
Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.
Subject(s)
Myosin Heavy Chains/chemistry , Nanotechnology , Biological Transport , Humans , Mechanotransduction, Cellular , Myosin Heavy Chains/ultrastructure , Optical ImagingABSTRACT
Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.
Subject(s)
Cytoplasmic Dyneins/metabolism , DNA/chemistry , DNA/metabolism , Kinesins/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Cytoplasmic Dyneins/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Kinesins/chemistry , Kymography , Molecular Motor Proteins/chemistry , Nucleic Acid Conformation , Protein Multimerization , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.
Subject(s)
Actomyosin/chemistry , Adenosine Triphosphatases/metabolism , Myosins/chemistry , Actins/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Division , Cloning, Molecular , Cross-Linking Reagents/pharmacology , Cysteine/chemistry , Dictyostelium , Disulfides , Electrophoresis, Polyacrylamide Gel , Models, Molecular , Mutagenesis , Mutagenesis, Site-Directed , Mutation , Myosins/genetics , Myosins/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Time Factors , Transformation, GeneticABSTRACT
The molecular motor myosin is proposed to bind to actin and swing its light-chain binding region through a large angle to produce an approximately 10 nm step in motion coupled to changes in the nucleotide state at the active site. To date, however, direct dynamic measurements have largely failed to show changes of that magnitude. Here, we use a cysteine engineering approach to create a high resolution, FRET-based sensor that reports a large, approximately 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.
