Thomas J. Purcell

Myosin VI is a processive motor with a large step size

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.

Role of the lever arm in the processive stepping of myosin V

Myosin V is a two-headed molecular motor that binds six light chains per heavy chain, which creates unusually long lever arms. This motor moves processively along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed myosin V tightly binds to actin and swings its long lever arm through a large angle, providing a stroke. We created single-headed constructs with different-size lever arms and show that stroke size is proportional to lever arm length. In a two-headed molecule, the stroke provides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processive step seen in tissue-purified myosin V. Two-headed myosin V molecules with only four light chains per head are still processive, but their step size is reduced to 24 nm. A further reduction in the length of the lever arms to one light chain per head results in a motor that is unable to walk processively. This motor produces single small ≈6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a given binding event. These data show that for myosin V with its normal proximal tail domain, both heads and a long lever arm are required for large, processive steps.

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase–nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.

Building and using optical traps to study properties of molecular motors.

Publisher Summary This chapter describes the building and using of optical traps to study the properties of molecular motors. Optical trapping experiments very directly measure the velocity, force, step size, and processive run length of molecular motors. The velocity, stall force, step size, and processive run length of molecular motors have all been determined using in vitro motility assays, both at the multiple- and the single-molecule levels. Optical trapping experiments in particular are used to gain information on stall forces, step sizes, and kinetics of a variety of molecular motors, such as kinesins, myosins, and processive DNA enzymes. Optical trapping assays are now performed on a variety of different systems using experimental techniques that may apply well to new, uncharacterized molecular motors and other biological macromolecules as well. These developments in both the technology and the assays involved in optical trapping have made the technique more accessible, increasing the likelihood that the optical trap can become a standard microscope used in laboratories that study movement and force in biological systems.

Optical Traps to Study Properties of Molecular Motors

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

Nucleotide Pocket Thermodynamics Measured by EPR Reveal How Energy Partitioning Relates Myosin Speed to Efficiency

We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH(0) and ΔS(0), even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.

Combining EPR with Fluorescence Spectroscopy to Monitor Conformational Changes at the Myosin Nucleotide Pocket

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WTSLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin myosin diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins.

The Optical Trapping Dumbbell Assay for Nonprocessive Motors or Motors That Turn around Filaments

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

Attachment of anti-GFP antibodies to microspheres for optical trapping experiments.

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

Mechanics of Unconventional Myosins

Publisher Summary This chapter discusses the mechanics of unconventional myosins. The quantitative in vitro assays to measure the essential function of myosin and its ATP-driven movement along actin are discussed in the chapter. These quantitative in vitro motility assays provide the evidence needed to rule out some models of contraction, such as those involving conformational changes in the tail region of the molecule. The cross-bridge itself (subfragment 1 of myosin or S1) is directly and unequivocally the motor domain of myosin. The tail of myosin II has the critical function of forming bipolar thick filaments that anchor the S1 motor domains to a macromolecular assembly used in muscle contraction and in contractile processes, such as cytokinesis in nonmuscle cells. The determination of the high-resolution crystal structures of S1 from the conventional myosin II show that it consists of a catalytic domain and a distinct light-chain binding domain. The catalytic domains contain a nucleotide-binding site of the P-loop variety that is closely associated with switch I and switch II helices. The nucleotide site is ∼ 4 nm away from the actin-binding site, and these two sites communicate with one another via the switch I and switch II helices, which move in response to the state of the nucleotide, especially the presence or absence of phosphate (Pi) in the active site.