Plasma formation in holmium:YAG laser lithotripsy.

Objectives: During holmium:yttrium-aluminum-garnet (holmium:YAG) laser lithotripsy to break urinary stones, urologists frequently see flashes of light. As infrared laser pulses are invisible, what is the source of light? Here we studied the origin, characteristics, and some effects of flashes of light in laser lithotripsy.

Methods: Ultrahigh-speed video-microscopy was used to record single laser pulses at 0.

High-speed video microscopy and numerical modeling of bubble dynamics near a surface of urinary stone.

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.

Loop L5 assumes three distinct orientations during the ATPase cycle of the mitotic kinesin Eg5: a transient and time-resolved fluorescence study.

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states.

The structural basis of force generation by the mitotic motor kinesin-5.

Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement.

A universal pathway for kinesin stepping.

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 the 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.

Loop L5 acts as a conformational latch in the mitotic kinesin Eg5.

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an α-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 nucleotide-induced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker.

The ATPase cycle of the mitotic motor CENP-E.

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding.

Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid.

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.

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.

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 high-resolution optical trap using a unique force clamp arrangement with fast response times.