Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.

The motor protein dynein is predicted to move the tail domain, a slender rod-like structure, relative to the catalytic head domain to carry out its power stroke. Here, we investigated ATP hydrolysis cycle-dependent conformational dynamics of dynein using fluorescence resonance energy transfer analysis of the dynein motor domain labeled with two fluorescent proteins. We show that dynein adopts at least two conformational states (states I and II), and the tail undergoes ATP-induced motions relative to the head domain during transitions between the two states. Our measurements also suggest that in the course of the ATP hydrolysis cycle of dynein, the tail motion from state I to state II takes place in the ATP-bound state, whereas the motion from state II to state I occurs in the ADP-bound state. The latter tail motion may correspond to the predicted power stroke of dynein.

A TP synthase of mitochondria, chloroplasts, and bacteria catalyzes ATP synthesis coupled with a transmembrane proton flow (1-4). The enzyme consists of a membraneembedded, proton-conducting portion (F 0 ) and a protruding portion (F 1 ) in which catalytic sites for ATP synthesis͞hydrolysis exist. The isolated F 1 portion has ATPase activity; hence, it is often called F 1 -ATPase. It is composed of five different subunits with a stoichiometry of ␣ 3 ␤ 3 ␥␦. The ␣ 3 ␤ 3 ␥ subcomplex is the minimum ATPase-active complex, which has catalytic features similar to F 1 -ATPase. In the crystal structure (5), the central ␥ subunit is surrounded by an ␣ 3 ␤ 3 cylinder where three ␣ and three ␤ subunits are arranged alternately, and the six nucleotide binding sites are located at the ␣͞␤ subunit interfaces. Three of the binding sites are catalytic, and the ␤ subunits provide most of the catalytic residues. The other three are noncatalytic, and the ␣ subunits provide most residues contributing nucleotide binding.It has been postulated that the energy of the proton flow liberated at F 0 is transformed into the energy of ATP synthesis at F 1 through rotation of the central ␥ subunit and vice versathe energy of ATP hydrolysis can be converted into the energy of proton pumping through reverse rotation of the ␥ subunit (6). By using an ␣ 3 ␤ 3 ␥ subcomplex of thermophilic F 1 -ATPase (F 1 -ATPase) immobilized on a glass surface, we have observed ATP hydrolysis-driven rotation of the fluorescent actin filament attached to the ␥ subunit (7).At nanomolar ATP concentration, F 1 -ATPase binds and hydrolyzes a single ATP molecule, makes a 120°rotation, and waits for the next ATP molecule. As the ATP concentration increases, the ATP-waiting period becomes shorter until it is finally undetectable, and rotation of the actin filament becomes apparently continuous over hundreds of revolutions (8). However, when the rotation was observed for long periods, occasional pauses of rotation were recognized, even at high ATP concentrations (7, 9). Here, we show that these pauses occur at an intermediate step of rotation and mostly correspond to the ADP-Mg inhibition, which has been observed in bulk-phase kinetics as a general feature of the F 1 -ATPases (and ATP synthases). Slow interconversion between rotating and pausing states thus contributes to the attenuation of ATPase during steady-state catalysis. Materials and MethodsProtein Preparation. Escherichia coli strains used were JM109 (10) for preparation of plasmids, CJ236 (11) for generating uracilcontaining single-stranded plasmids for site-directed mutagenesis, and JM103⌬ (uncB-uncD) for expression of the mutant complexes of F 1 from the thermophilic Bacillus PS3. Plasmids M13mp18 and pKAGB1 (12), which carried genes for the ␣, ␤, and ␥ subunits of F 1 from the thermophilic Bacillus PS3, were used for mutagenesis and for gene expression, respectively. Site-directed mutagenesis was accomplished as described by Kunkel et al. (11). The plasmid pKAGB1͞␣C193S͞␥S107C͞ ␤His10tag has been described ...

We have established an orientation technique of microtubules and evaluated their polarities by the movement of kinesin-coated beads quantitatively in poly(dimethyl siloxane) (PDMS) channels. More than 95% of beads moved to the desired direction; this indicates almost all the microtubules were functionally oriented including plus and minus polarities. The technique is essential for fabricating a bio-hybrid nanotransport system, in which the kinesin−microtubule system combined with microfluidic structures provides a driving mechanism in an aqueous environment: once microtubules are immobilized inside a microfluidic channel, kinesin-coated objects can be transported on the designated pathways without any liquid manipulation.

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