Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.

Recent developments in piconewton instrumentation allow the manipulation of single molecules and measurements of intermolecular as well as intramolecular forces. Dextran filaments linked to a gold surface were probed with the atomic force microscope tip by vertical stretching. At low forces the deformation of dextran was found to be dominated by entropic forces and can be described by the Langevin function with a 6 angstrom Kuhn length. At elevated forces the strand elongation was governed by a twist of bond angles. At higher forces the dextran filaments underwent a distinct conformational change. The polymer stiffened and the segment elasticity was dominated by the bending of bond angles. The conformational change was found to be reversible and was corroborated by molecular dynamics calculations.

The rupture force of single covalent bonds under an external load was measured with an atomic force microscope (AFM). Single polysaccharide molecules were covalently anchored between a surface and an AFM tip and then stretched until they became detached. By using different surface chemistries for the attachment, it was found that the silicon-carbon bond ruptured at 2.0 +/- 0.3 nanonewtons, whereas the sulfur-gold anchor ruptured at 1.4 +/- 0.3 nanonewtons at force-loading rates of 10 nanonewtons per second. Bond rupture probability calculations that were based on density functional theory corroborate the measured values.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

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Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete Ϸ36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity. C lass-V myosins, two-headed actin-based motors (1), have been implicated in several forms of organelle transport (2). The various roles of molecular motors require special kinetic adaptations (3). Unlike muscle myosin-II, which assembles in large arrays, myosin-V is a processive motor (4), meaning that one molecule can undergo multiple productive catalytic cycles and associated mechanical steps before it detaches from its track. To understand the mechanism for chemomechanical transduction, one must decipher the kinetic scheme underlying ATP turnover and movement. Presteady-state kinetic studies have helped clarify such mechanisms in many motor proteins (5). In the case of myosin-V, kinetic characterization of truncated single-headed constructs in bulk studies has contributed important insights into the myosin-V ATPase cycle (6, 7). However, to understand the mechanism for myosin-V processivity, it is essential to study the full-length double-headed dimer through the course of its movement. In the present study, we used a force feedback-enhanced laser trap to measure the stepping rate of myosin-V molecules purified from brain. This allowed us to characterize the rate-limiting transition in the turnover cycle. Materials and MethodsBead Preparation. One-microliter Polystyrene beads (Ø 356 nm, Polysciences, 2.5% solid) were incubated for 15 min in 99 l of buffer (25 mM imidazole HCl, pH 7.4͞25 mM KCl͞1 mM EGTA͞10 mM DTT͞4 mM MgCl 2 ) containing 10 g͞ml BSA (to preblock the surface), 1 g͞ml tetramethyl rhodaminelabeled BSA, and 30 pM tissue-purified chick-brain myosin-V [purification as described in (8) Optical Trap. Beads were optically trapped and positioned near a fluorescently labeled biotinylated actin filament immobilized onto an avidin-coated coverslip. Imaging and trap steering were as described (9-11). A feedback loop (M44 DSP-board, Innovative Integration, West Lake Village, CA) maintains a constant separation between the bead and trap centers. This distance scales with the load experienced by the molecule as it steps along the actin filament. The trap stiffness was calibrated for each trapped bead from the amplitude of the thermal diffusion. For some beads, it was also calibrated by measurement of the bead rise time in response to sudden trap displacement and by the 3-dB corner frequency in the diffusion power spectrum. The three methods gave consistent results. Results and DiscussionPolystyrene beads, sparsely coated with myosin-V molecules, were optically trapped ...