In examining the mechanism of rotary motors, we have primarily chosen to focus on helicases and FoF1-ATPase. Several models have been proposed to explain precisely how helicases separate both strands of DNA, which is somewhat similar to the way a zipper works. Most consistent with the existing data is the wedge model, in which NTPase activity allows the helicase to move unidirectionally along a strand of dsDNA and separate it from the other strand. In the torsional model, one strand is bound in the central channel of the helicase and the other is bound to the outside of the helicase; this action generates torque which unwinds the two strands. The helix-destabilizing model is similar to the torsional model except protein–DNA interactions and conformational changes separate the two strands, rather than torque. The process by which helicases translocate nucleic acid and unwind the DNA duplex is tied to cycle of NTP binding and hydrolysis. We then look at each of the two complex motors of FoF1-ATPase individually. ATP, the basis for cell energy in all plants, animals, and microorganisms, is synthesized from ADP and inorganic phosphate and is generated by FoF1-ATPase. Single-molecule rotation assay has provided direct evidence of ATP-driven rotation in the F1 motor. In several kinds of rotary ATPases, F1 is seen to rotate the γ subunit in distinct 120° counterclockwise steps with a single turnover of ATP hydrolysis. Each step can be divided into two substeps: an 80° substep driven by ATP binding and ADP release on different β subunits, and a 40° substep that begins after the release of ADP and inorganic phosphate. Unlike F1, which is powered by ATP, force is generated in Fo by proton flow, with the Fo in some bacteria fueled by sodium motive force. While the complexity of the structure of Fo is not well known, a number of models exist that examine the working principles of this motor. The most widely accepted model for proton translocation in Fo is the two-channel model, which posits that there are two hemichannels in the a subunit, each of which interact with the ion-conducting carboxyl residue of two c subunits. A proton enters the a subunit and, after being transferred to the c subunit, the released proton enters the cytoplasmic space, accompanied by one clockwise rotation of the c-ring per proton. When the process is driven by ATP rather than proton flow, the process is reversed, resulting in counterclockwise rotation. Exploration of the mechanisms behind the rotation of the c-ring is a key element in understanding how the Fo motor operates.
The way helicases, a type of rotary motor, separate the two strands of DNA is somewhat similar to how the metal piece on a zipper separates both rows of teeth and unzips them. Like a zipper, the helicase creates a y-shaped fork where the two strands of DNA part known as a replication fork, ahead of which the helicase travels. While the process by which helicases unwind dsDNA is not fully understood, several models have been proposed. Most consistent with the existing data is the wedge model. One strand of the dsDNA is bound in the helicase channel, and NTPase activity permits the helicase to move unidirectionally along it, excluding the other strand and destabilizing the bond between them in the manner of a wedge. In the torsional model, both of the strands interact with the helicase, with one bound tightly in the central channel and the other bound to the outer part of the helicase. This generates torque and rotates one of the strands, allowing them to unwind. The helix-destabilizing model is similar to the torsional model in which the helicase interacts with both strands, one bound in the central channel and the other to the outer part. The only difference is that the protein–DNA interactions and conformational changes caused by NTPase activity separate the base pairing, and the helicase moves unidirectionally along the strand that is bound in the central channel. The speed of the DNA helicase is incredibly fast: It can turn at a maximum of 10,000 rotations per minute, on par with a jet engine turbine.
Nucleic acid translocation and duplex unwinding by helicases are coupled to the NTP-binding and -hydrolysis cycle. In nucleic acid translocation, there are six NTP-binding sites, as well as at least six nucleic acid-binding sites in the hexamer. Individual subunits of a hexameric helicase may switch between several DNA-binding states. The translocation model is based upon the notion of a fluctuating electrostatic field (Doering et al., 1998). According to this model, the binding of NTP to a hydrolysis site near the inner surface of the channel induces a conformational change that exposes one pair of negatively and positively charged regions per nucleotide-hydrolysis site. These charged regions are not of equal size, are oriented at an angle to the circumferential meridian, and are not constant, appearing and disappearing with the binding and hydrolysis of NTP, respectively. The field of these charged regions can sequentially interact with the closest negatively charged DNA phosphate, which gives an “electrostatic push” in the direction of the charged pair axis. The combined effect of the charged pairs produces a sustained torsional and axial thrust.
In FoF1 synthase, binding of ADP and inorganic phosphate forms a phosphate–phosphate bond, causing ATP to be synthesized or hydrolyzed. If the ATP concentration is high and the electrochemical potential low, ATP hydrolysis drives the stronger F1 motor to cause the Fo motor to reverse and creates a proton pump. F1 has been directly observed in an optical microscope to move in distinct 120° steps, each of which is powered by the hydrolysis of one ATP molecule, composed of 90° and 30° substeps driven by ATP binding and ADP release, respectively.