Session G43: Focus Session: Motor dynamics---from Single Molecules to Cells II

Operation mechanism of rotary molecular motor F$_1$ probed by single-molecule techniques

F$_1$ is a rotary motor protein. Three catalytic $\beta $-subunits in the stator $\alpha_{3}\beta_{3}$ ring are torque generators, and rotate the rotor $\gamma $-subunit by sequential and cooperative conformational changes coupled with adenosine triphosphate (ATP) hydrolysis reaction. F$_1$ shows remarkable performances such as rotation rate faster than 10,000 rpm, high reversibility and efficiency in chemo-mechanical energy conversion. I will introduce basic characteristics of F$_1$ revealed by single-molecule imaging and manipulation techniques based on optical microscopy and high-speed atomic force microscopy. I will also discuss the possible operation mechanism behind the F$_1$, along with structurally-related hexameric ATPases, also mentioning the possibility of generating hybrid molecular motors.   

Simultaneous measurement of DNA motor protein conformation and activity with combined optical trap and single-molecule fluorescence

We present single-molecule measurements of Superfamily 1 UvrD helicase DNA unwinding that reveal directly how helicase stoichiometry and conformation regulate motor activity. Using a new instrument that combines high resolution optical tweezers with single-molecule fluorescence microscopy, we record DNA unwinding activity with base pair-scale resolution (via optical tweezers) simultaneously with helicase stoichiometry and conformation (via fluorescence). Quantifying the fluorescence signal from labeled UvrD, we observe that pairs of UvrD molecules are required for long distance unwinding but that individual molecules exhibit limited, non-processive unwinding activity. UvrD is also known to exhibit two different conformations, `closed' and `open', based on the orientation of its 2B regulatory domain. The function of these conformations has remained elusive. Measuring the fluorescence of FRET labeled proteins, we detect directly the conformation of the 2B domain of individual UvrD molecules during unwinding activity. We observe that UvrD is in the `closed' conformation during DNA unwinding but surprisingly switches to the `open' conformation upon reversal of helicase direction, i.e. when UvrD switches strands and translocates on the opposing strand with the DNA junction rezipping behind it. We hypothesize that the 2B domain acts as a conformational switch that controls DNA unwinding vs. re-annealing.   

How Interactions Affect Multiple Kinesin Dynamics

Intracellullar transport is supported by several classes of enzymatic molecules known as motor proteins. Cellular cargos are frequently transported by teams of motor proteins, and recent experimental and theoretical studies uncovered many features of such complex dynamics. Here we investigate theoretically the role of nonmechanical interactions between kinesin motor proteins and microtubules in the collective motion of motor proteins. Our analysis is based on stochastic model that explicitly takes into account all chemical and mechanical transitions. Nonmechanical interactions are assumed to affect kinesin mechanochemistry only when the motors are separated by less than 3 microtubule lattice sites, and it is shown that relatively weak interaction energies can have a significant effect on collective motor dynamics. In agreement with optical trapping experiments on structurally defined kinesin complexes, the model predicts that these effects primarily occur when cargos are transported against loads exceeding single-kinesin stalling forces. These results highlights the complex dynamics of multiple motor proteins in cellular transport phenomena.   

Molecular mechanism of motion and force generation by cytoplasmic dynein

Cytoplasmic dynein is an intricate microtubule (MT) motor with four AAA (ATPase associated with various cellular activities) ATPases per head domain. Dynein homodimers take hundreds of consecutive steps, during which the leading and trailing heads experience intramolecular tension in opposite directions. We hypothesize that this asymmetry may differentially regulate the MT-binding and ATPase functions in each head, thereby facilitating processive movement. Here, we elucidate the function of tension in regulating dynein-MT interactions, and dissect the roles of its multiple AAA subunits in effecting and modulating this behavior. Using optical tweezers to measure unbinding forces of single S. cerevisiae dynein heads in the absence of nucleotide, we show that intrinsic dynein-MT binding is significantly weaker under forward (MT-minus-end directed) tension than under rearward tension. Thus, forward tension likely promotes rear head detachment in the dimeric motor. The nucleotide states of specific AAA sites modify this intrinsic behavior. Mutational analysis shows that ATP binding to AAA1 substantially weakens MT binding. Moreover, ADP binding to AAA3 `locks' dynein in a previously undescribed, weak MT-binding state with a relatively symmetric response to tension. Interestingly, tension also affects nucleotide affinity: ADP affinity is lower under rearward than under forward load, suggesting that the front head preferentially releases ADP (likely from AAA3), perhaps driving a transition from an ADP state with relatively weak MT attachment to a strongly MT-attached, nucleotide-free state. Our analysis suggests that intramolecular tension is key to dynein motility, and highlights the importance of including multiple AAA ATPases in models for dynein mechanochemistry.   

Motor-motor interactions in ensembles of muscle myosin: using theory to connect single molecule to ensemble measurements

Interactions between the proteins actin and myosin drive muscle contraction. Properties of a single myosin interacting with an actin filament are largely known, but a trillion myosins work together in muscle. We are interested in how single-molecule properties relate to ensemble function. Myosin's reaction rates depend on force, so ensemble models keep track of both molecular state and force on each molecule. These models make subtle predictions, e.g. that myosin, when part of an ensemble, moves actin faster than when isolated. This acceleration arises because forces between molecules speed reaction kinetics. Experiments support this prediction and allow parameter estimates. A model based on this analysis describes experiments from single molecule to ensemble. In vivo, actin is regulated by proteins that, when present, cause the binding of one myosin to speed the binding of its neighbors; binding becomes cooperative. Although such interactions preclude the mean field approximation, a set of linear ODEs describes these ensembles under simplified experimental conditions. In these experiments cooperativity is strong, with the binding of one molecule affecting ten neighbors on either side. We progress toward a description of myosin ensembles under physiological conditions.