Craig L. Hetherington

High Degree of Coordination and Division of Labor among Subunits in a Homomeric Ring ATPase

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

Mechanochemistry of a Viral DNA Packaging Motor

The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P(i) release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage phi12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA decreases towards the end of the power stroke and the DNA rolls to the lever on the next subunit. Each power stroke facilitates ATP hydrolysis in the next catalytic site by inserting an Arg -finger into the site, as captured in phi12-P4. At the end of every four power strokes, ADP release happens slowly, so the cycle pauses constituting a dwell phase during which four ATPs are loaded into the catalytic sites. The next burst phase of four power strokes starts once spontaneous ATP hydrolysis takes place in the fifth site without insertion of an Arg finger. The push and roll model provides a new perspective on how a multimeric ATPase transports DNA, and it might apply to other ring motors as well.

A Viral Packaging Motor Varies Its DNA Rotation and Step Size to Preserve Subunit Coordination as the Capsid Fills

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motors step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.

Origin of Reversible Photoinduced Phase Separation in Hybrid Perovskites

The distinct physical properties of hybrid organic-inorganic materials can lead to unexpected nonequilibrium phenomena that are difficult to characterize due to the broad range of length and time scales involved. For instance, mixed halide hybrid perovskites are promising materials for optoelectronics, yet bulk measurements suggest the halides reversibly phase separate upon photoexcitation. By combining nanoscale imaging and multiscale modeling, we find that the nature of halide demixing in these materials is distinct from macroscopic phase separation. We propose that the localized strain induced by a single photoexcited charge interacting with the soft, ionic lattice is sufficient to promote halide phase separation and nucleate a light-stabilized, low-bandgap, ∼8 nm iodide-rich cluster. The limited extent of this polaron is essential to promote demixing because by contrast bulk strain would simply be relaxed. Photoinduced phase separation is therefore a consequence of the unique electromechanical properties of this hybrid class of materials. Exploiting photoinduced phase separation and other nonequilibrium phenomena in hybrid materials more generally could expand applications in sensing, switching, memory, and energy storage.

Peptide nucleic acids as tools for single-molecule sequence detection and manipulation.

The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers.

4.22 Viral DNA Packaging Motors

In viral DNA packaging, the genome is compacted to high densities into a capsid by an ATP-hydrolyzing molecular motor. The study of viral packaging has advanced dramatically in recent years due to new biophysical methods, particularly single-molecule techniques. We review the insights afforded by these new tools into the initiation, translocation, and termination phases of the packaging process. Furthermore, by integrating single-molecule data with structural and biochemical information from related molecular motors, we develop and present a possible molecular model for ATP-dependent translocation by a viral packaging motor.

Noninvasive Cathodoluminescence-Activated Nanoimaging of Dynamic Processes in Liquids

In situ electron microscopy provides remarkably high spatial resolution, yet electron beam irradiation often damages soft materials and perturbs dynamic processes, requiring samples to be very robust. Here, we instead noninvasively image the dynamics of metal and polymer nanoparticles in a liquid environment with subdiffraction resolution using cathodoluminescence-activated imaging by resonant energy transfer (CLAIRE). In CLAIRE, a free-standing scintillator film serves as a nanoscale optical excitation source when excited by a low energy, focused electron beam. We capture the nanoscale dynamics of these particles translating along and desorbing from the scintillator surface and demonstrate 50 ms frame acquisition and a range of imaging of at least 20 nm from the scintillator surface. Furthermore, in contrast with in situ electron microscopy, CLAIRE provides spectral selectivity instead of relying on scattering alone. We also demonstrate through quantitative modeling that the CLAIRE signal from metal nanoparticles is impacted by multiplasmonic mode interferences. Our findings demonstrate that CLAIRE is a promising, noninvasive approach for super-resolution imaging for soft and fluid materials with high spatial and temporal resolution.

A Mechanochemical Model of a Viral DNA Packaging Motor

Packaging the genome of a virus into its capsid is a crucial step in viral assembly. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA) of about 19,000 base pairs (bps). Packaging a dsDNA this long results in a near-crystalline state inside the ∼50 nm length capsid and requires a great deal of energy. The feat is performed by a multimeric molecular motor that derives its energy from ATP hydrolysis and generates forces more than 60 pN. Experimental studies on the phi29 packaging motor have been carried out through single-molecule manipulation techniques using optical tweezers. The DNA packaging proceeds in bursts of four 2.5-bp translocation power strokes upon Pi releases. The translocation is also accompanied by the DNA rotation. From the data we have constructed a mechanochemical framework to explain how this motor packages DNA. The model is built around ‘push-and-roll’mechanism that suggests how the motor subunits interact with the DNA and how the DNA passes through the motor ring. We also propose how the five subunits are coordinated around the ring. Our model provides a new perspective on how multimeric ATPases transport nucleic-acids, and it may be applied to other ring motors.