Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a highresolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6 -30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.DNA hairpin ͉ energy landscape ͉ force clamp ͉ optical tweezers ͉ single molecule H airpins formed from self-complementary sequences supply a model system for studying folding and duplex formation, the most fundamental processes for generating structure in nucleic acids. Using hairpins, repeated measurements can be made on the same molecule, facilitating single-molecule studies. Furthermore, by simply altering the nucleotide sequence, physical properties such as folding energies, kinetic rates, and distances to transition states all can be changed systematically. Hairpins also play essential roles in vivo. DNA hairpins bind proteins to regulate transcription (1), and hairpin intermediates are involved in both replication and recombination (2, 3). RNA hairpins form tertiary contacts (4), bind to proteins (2), regulate transcription (5), and mediate RNA interference (6). Understanding the factors that influence hairpin folding should therefore not only elucidate principles of structure formation in nucleic acids but may also shed light on the biological roles played by these structures.Extensive calorimetric and melting studies have been carried out to generate predictive rules for the thermodynamic stability of arbitrary nucleic acid duplexes (7). The kinetic properties of duplex formation, however, remain less well understood, particularly those related to the nature of the transition state. Temperature-jump studies of annealing in short duplexes have been interpreted in terms of the nucleation of a transition state consisting of Ϸ1-3 bp, followed by zippering of the remaining stem (8). This interpretation, however, rests on the assumption that the enthalpy of activation arises ...

Nucleic acid hairpins provide a powerful model system for understanding macromolecular folding, with free-energy landscapes that can be readily manipulated by changing the hairpin sequence. The full shapes of energy landscapes for the reversible folding of DNA hairpins under controlled loads exerted by an optical force clamp were obtained by deconvolution from high-resolution, single-molecule trajectories. The locations and heights of the energy barriers for hairpin folding could be tuned by adjusting the number and location of G:C base pairs, and the presence and position of folding intermediates were controlled by introducing single-nucleotide mismatches.

Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Due to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state where both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. Based on measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we find that neck-linker orientation is also responsible for ensuring gating in kinesin.

Background: Kinesin-5 motors are important for formation and maintenance of the bipolar mitotic spindle. Results: ATP binding triggers coupled conformational changes of kinesin-5 specific structural elements in the microtubulebound motor domain. Conclusion: Kinesin-5 mechanochemistry is tuned to its cellular functions. Significance: Subnanometer resolution structure determination of microtubule-bound kinesin-5s and kinetics experiments reveal the molecular basis of their motor properties and of drug inhibition.

Ultra-high-speed video microscopy and numerical modeling were used to assess the dynamics of microbubbles at the surface of urinary stones. Lipid-shell microbubbles designed to accumulate on stone surfaces were driven by bursts of ultrasound in the sub-MHz range with pressure amplitudes on the order of 1 MPa. Microbubbles were observed to undergo repeated cycles of expansion and violent collapse. At maximum expansion, the microbubbles' cross-section resembled an ellipse truncated by the stone. Approximating the bubble shape as an oblate spheroid, this study modeled the collapse by solving the multicomponent Euler equations with a two-dimensional-axisymmetric code with adaptive mesh refinement for fine resolution of the gas-liquid interface. Modeled bubble collapse and high-speed video microscopy showed a distinctive circumferential pinching during the collapse. In the numerical model, this pinching was associated with bidirectional microjetting normal to the rigid surface and toroidal collapse of the bubble. Modeled pressure spikes had amplitudes two-to-three orders of magnitude greater than that of the driving wave. Micro-computed tomography was used to study surface erosion and formation of microcracks from the action of microbubbles. This study suggests that engineered microbubbles enable stone-treatment modalities with driving pressures significantly lower than those required without the microbubbles.

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an ␣-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotideinduced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with ␣3 and in so doing controls the speed of movement and force generation in kinesin motors.The kinesin superfamily of molecular motors share several evolutionarily conserved structural features with myosins and G-proteins. These include domains that coordinate the ␤ phosphate of bound nucleotide (P loop), that sense the ␥ phosphate (switch I), and that induce conformational changes in response to phosphate release (switch II) (1-3). However, kinesins also contain an unusual structural element not found in these other motors and switches. This consists of an ␣-helix (␣2) on the carboxyl terminal end of the P loop, which is interrupted by a stem and loop motif referred to as L5 (Ref. 4 and Fig. 1). The length of L5 varies considerably between different kinesin superfamily members, from short in kinesin 1 and CENP-E to longest in the mitotic kinesin Eg5 (Fig. 2).The function of L5 remains unclear, although three lines of evidence suggest that it may play important roles in regulating nucleotide and microtubule binding. First, several small molecules induce L5 in Eg5 to fold over, generating a hydrophobic pocket bounded by L5, ␣2, and ␣3 (5, 6). In this conformation both ADP release and microtubule binding are prevented (7,8). The analogous site in CENP-E is also the binding site for a novel inhibitor stabilizing the microtubule-motor complex, which appears to stabilize this motor in a strong microtubule binding conformation (9). Second, labeling of L5 with an EPR spin probe demonstrates that its mobility is affected by nucleotide binding, and in turn, deletion of L5 affects nucleotide binding (10). Third, cryo-EM reconstructions of Eg5-decorated microtubules in the presence of AMPPNP suggests that L5 can interact with ␣3, when the Eg5 motor is bound to the microtubule (11). Because the ␣3 helix is on the amino-terminal side of switch I, changes in the L5-␣3 interaction might have downstream effects on nucleotide affinity and, secondarily, on microtubule binding. However, this study generated a docking model using crystallographic structures of Eg5 in the p...

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Double-stranded DNA viruses, including tailed bacteriophages and mammalian herpesviruses, package their genomes into pre-formed protein capsids. The packaging process is driven by a molecular complex known as the packaging motor. This motor is a ring-shaped oligomeric ATPase that utilizes the chemical energy from ATP binding and hydrolysis to perform the mechanical work. We have recently presented a detailed mechanochemical characterization for the bacteriophage phi29 motor, a homo-pentamer that translocates DNA in cycles composed of alternating dwells and bursts (1). We now aim to investigate the behavior of the motor at different stages of packaging, as the motor needs to overcome increasing amounts of internal pressure generated by the compressed dsDNA. We find that the effect of the internal pressure on the motor dynamics is more complex than simply exerting an opposing force to packaging. Through a detailed analysis using ultra-high-resolution DNA translocation data, we find that the internal pressure affects multiple kinetic transitions in the mechanochemical cycle, including events in both the dwell phase and the burst phase. This analysis allows us to make an accurate estimation of the internal force as a function of DNA filling, which is important for the understanding of the DNA organization inside the capsid and the ejection energetics. Remarkably, the motor changes its step size and displays a new class of long-lived pauses towards the completion of packaging. Finally, we determine the structural elements in the packaging complex that are responsible for the signal transduction from the capsid to the motor.(1) In this work, we explored the possible existence of a wear mechanism developing under mild conditions using surface force apparatus (SFA). By selectively digesting different components (type II collagen, Hyaluronic acid (HA), glycosaminoglycans (GAGs)) of the cartilage, we also establish correlation between the structural properties of cartilage surface and the tribological properties. We observed stick-slip friction in articular cartilage for the first time under mild conditions. To visualize load and speed regimes of stick-slip friction occurrence we introduced 'Dynamic (friction) phase diagram'. Prolonged exposure of the cartilage surfaces to stick-slip sliding results in an increase of surface roughness similar to HA and GAGs digested cartilage suggesting that stick-slip motion is able to induce severe morphological changes of cartilage superficial zone. We also found that digestion of the different components of cartilage alters the morphology of the superficial zone in very different ways. HA and GAGs digestions increased surface roughness while collagen digestion decreased the surface roughness compared to normal cartilage. Friction forces increased up to 2, 5 and 10 times after HA, collagen and GAGs digestion, respectively, indicating no direct correlation between surface roughness and friction forces. Kinesin-5s are essential for forming the bipolar spindle during mitosis in most eukary...