Ultrafast Force-clamp Spectroscopy Research Articles

High-Speed Optical Traps Address Dynamics of Processive and Non-Processive Molecular Motors.

Interactions between biological molecules occur on very different time scales, from the minutes of strong protein-protein bonds, down to below the millisecond duration of rapid biomolecular interactions. Conformational changes occurring on sub-ms time scales and their mechanical force dependence underlie the functioning of enzymes (e.g., motor proteins) that are fundamental for life. However, such rapid interactions are beyond the temporal resolution of most single-molecule methods. We developed ultrafast force-clamp spectroscopy (UFFCS), a single-molecule technique based on laser tweezers that allows us to investigate early and very fast dynamics of a variety of enzymes and their regulation by mechanical load. The technique was developed to investigate the rapid interactions between skeletal muscle myosin and actin, and then applied to the study of different biological systems, from cardiac myosin to processive myosin V, microtubule-binding proteins, transcription factors, and mechanotransducer proteins. Here, we describe two different implementations of UFFCS instrumentation and protocols using either acousto- or electro-optic laser beam deflectors, and their application to the study of processive and non-processive motor proteins.

Erratum: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy.

An erratum was issued for:Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy. The title was updated. The title was updated from: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy to: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy.

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy.

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force. By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast Force-Clamp Studies of the Diffusing Microtubule-Binding Proteins

Chromosome segregation during cell division crucially depends on the activities of the microtubule associated proteins (MAPs) that couple chromosomal kinetochores to spindle microtubules. When a kinetochore-bound microtubule shortens, it pulls the coupled kinetochore toward the spindle pole-associated microtubule minus-end, whereas the sister kinetochore is dragged toward the microtubule plus-end. However, there is little understanding of how the coupling kinetochore MAPs translocate along microtubule in different directions under the dragging force. To bridge this gap, we applied a highly sensitive ultrafast force-clamp spectroscopy to investigate directional mobility of key kinetochore components, including human Ndc80 and Ska complexes, and the ring-forming Dam1 complex from yeast. Single molecules of these proteins were conjugated to the coverslip-immobilized bead pedestals and the microtubule “dumbbell” was oscillated near the pedestal's surface under a continuously operating force clamp. Surprisingly, the character of the force-induced translocation was dramatically different for these MAPs, even though they undergo thermal diffusion along microtubules at highly similar rates. Under the dragging force, the Ska complex glides smoothly in both microtubule directions, whereas the Ndc80 exhibits differential gliding and the Dam1 shows asymmetric force-induced static bindings. These results strongly support the model in which the translocating kinetochore MAPs function as integral components of the molecular machine powered by microtubule depolymerization, rather than by biased diffusion. We propose that the asymmetric behavior of Ndc80 and Dam1 complexes underlies differential internal friction at moving kinetochores, which enables their poleward-directed gliding, while reducing the microtubule end slippage under tension.

Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Coupling of Bond Breaking With State Transition Leads to High Apparent Detachment Rates of a Single Myosin

Quantifying interactions between motors and filaments is important for the understanding of intriguing emergent behaviors of motor–filament systems, which play critical roles in various biological processes. Recently, unusually high detachment rates of a myosin from actin were obtained with a force spectroscopy technique of an unprecedented spatial–temporal resolution. Here, we suggest that these high apparent detachment rates may be due to the inherent coupling between bond breaking and state transition, which can be common in protein–protein interactions. Based on a kinetic model for the chemomechanical cycle of single myosin, rates of bond breaking between myosin and actin at different nucleotide states are systematically calculated. These quantitative results indicate that myosins may adopt much higher transition rates than bond breaking rates at different nucleotide states under physiological conditions when applied forces are relatively low. This work also indicates that accurate biophysical models considering both protein unbinding dynamics and protein state transitions are required in order to properly interpret the experimental data when the ultrafast force-clamp spectroscopy technique is employed to study, for example, the DNA–protein interactions.

Lac Repressor-DNA Interactions assessed by Ultrafast Force-Clamp Spectroscopy

We recently developed an ultrafast force-clamp laser trap technique [Capitanio et al., Nature Methods 9,1013-1019(2012)] that allows probing, under controlled force, both long- and short-lived biomolecular interactions (100μs to tens/hundreds of seconds), as well as sub-nanometer conformational changes occurring upon bond formation. Here, we show the application of our method to the study of lactose repressor (LacI). Our results show two kinetically well-distinct populations of interactions, which clearly represent strong interactions (targeting the two operators located 100nm apart from each other: long events in the figure) and fast scanning of LacI along non-cognate DNA (during target-search: short events in the figure). Our results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA molecule and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.This research is funded by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ultrafast Force-Clamp Spectroscopy of Single Molecular Motors and DNA Binding Proteins

Force plays a fundamental role in a wide array of biological processes, regulating, for example, enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. Here, we present a novel force-clamp laser trap that is capable of applying constant loads to molecular complexes with lifetimes above ∼10 μs [Capitanio et al., Nature Methods 9, 1013-1019 (2012)]. Such capability is enhanced by a detection strategy allowing the investigation of very short interactions (∼100μs). Moreover, the high temporal and spatial resolution of the method enables us to probe sub-nanometer conformational changes with a time resolution of few tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. These results resolve the molecular mechanism underlying regulation of muscle contraction by force. Our technique could be straightforwardly applied to a wide variety of non-processive molecular motors, single domains of processive motors, protein-DNA and protein-RNA interactions, and conceivably to any short-lived protein-protein interaction, opening new avenues for investigating the effects of forces on biological processes.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.