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.

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Mechanics of Single Vimentin Intermediate Filaments Under Load.

The eukaryotic cytoskeleton consists of three different types of biopolymers - microtubules, actin filaments, and intermediate filaments - and provides cells with versatile mechanical properties, combining stability and flexibility. The unique molecular structure of intermediate filaments leads to high extensibility and stability under load. With high laser power dual optical tweezers, the mechanical properties of intermediate filaments may be investigated, while monitoring the extension with fluorescence microscopy. Here, we provide detailed protocols for the preparation of single vimentin intermediate filaments and general measurement protocols for (i) stretching experiments, (ii) repeated loading and relaxation cycles, and (iii) force-clamp experiments. We describe methods for the analysis of the experimental data in combination with computational modeling approaches.

Miniaturized magnetic bead-actuators for force-clamp spectroscopy-based single-molecule measurements.

Force-clamp spectroscopy can mimic the physiological conditions for the proteins under investigation. In addition, it is a direct way of observing the relationship between bond lifetime and molecular forces. However, traditional force-clamp methods rely on active feedback controllers that can introduce artefacts. In this work, we introduce a new method to enable force-clamp spectroscopy without a need for an active feedback. The method is based on miniaturized magnetic beads offering improved stability. As a case study, we performed force-clamp experiments using biotin-streptavidin molecule pairs with and without active feedback. Our results demonstrate the feasibility of force-clamp experiments without feedback and illustrate the advantages of our method.

Data on the target search by a single protein on DNA measured with ultrafast force-clamp spectroscopy.

The mechanism by which proteins are able to find small cognate sequences in the range from few to few tens of base pairs amongst the millions of non-specific chromosomal DNA has been puzzling researchers for decades. Single molecule techniques based on fluorescence have been successfully applied to investigate this process but are inherently limited in terms of spatial and temporal resolution. We previously showed that ultrafast force-clamp spectroscopy, a single molecule technique based on laser tweezers, can be applied to the study of protein-DNA interaction attaining sub-millisecond and few base-pair resolution. Here, we share experimental records of interactions between a single lactose repressor protein and DNA collected under different forces using our technique [1]. The data can be valuable for researchers interested in the study of protein-DNA interaction and the mechanism of DNA target search, both from an experimental and modeling point of view. The data is related to the research article "Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching" [2].

Ultra-fast force-clamp spectroscopy data on the interaction between skeletal muscle myosin and actin.

Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers with sub-millisecond and sub-nanometer resolution. The technique has been successfully applied to investigate the rapid conformational changes that occur when a myosin II motor from skeletal muscle interacts with an actin filament. Here, we share data on the kinetics of such interaction and experimental records collected under different forces [1]. The data can be valuable for researchers interested in the mechanosensitive properties of myosin II, both from an experimental and modeling point of view. The data is related to the research article "ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke" [2].

Measuring Microtubule Supertwist and Defects by Three-Dimensional-Force-Clamp Tracking of Single Kinesin-1 Motors.

Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it is hypothesized that microtubule supertwist and defects can severely influence the function of motors and other microtubule-associated proteins, the presented method allows for the first time to fully map the microtubule lattice in situ. This mapping allows the correlation of motor-filament interactions with the microtubule fine-structure. With the additional ability to apply loads, we expect our 3D-optical-tweezers force clamp to become a valuable tool for obtaining a wide range of information from other biological systems, inaccessible by two-dimensional and/or ensemble measurements.

Single Molecule Studies Enabled by Model-Based Controller Design.

Optical tweezers have enabled important insights into intracellular transport through the investigation of motor proteins, with their ability to manipulate particles at the microscale, affording femto newton force resolution. Its use to realize a constant force clamp has enabled vital insights into the behavior of motor proteins under different load conditions. However, the varying nature of disturbances and the effect of thermal noise pose key challenges to force regulation. Furthermore, often the main aim of many studies is to determine the motion of the motor and the statistics related to the motion, which can be at odds with the force regulation objective. In this article, we propose a mixed objective H 2 /H ∞ optimization framework using a model-based design, that achieves the dual goals of force regulation and real time motion estimation with quantifiable guarantees. Here, we minimize the H ∞ norm for the force regulation and error in step estimation while maintaining the H 2 norm of the noise on step estimate within user specified bounds. We demonstrate the efficacy of the framework through extensive simulations and an experimental implementation using an optical tweezer setup with live samples of the motor protein 'kinesin'; where regulation of forces below 1 piconewton with errors below 10% is obtained while simultaneously providing real time estimates of motor motion.

The Nanomechanics of Lipid Multibilayer Stacks Exhibits Complex Dynamics.

The nanomechanics of lipid membranes regulates a large number of cellular functions. However, the molecular mechanisms underlying the plastic rupture of individual bilayers remain elusive. This study uses force clamp spectroscopy to capture the force-dependent dynamics of membrane failure on a model diphytanoylphosphatidylcholine multilayer stack, which is devoid of surface effects. The obtained kinetic measurements demonstrate that the rupture of an individual lipid bilayer, occurring in the bilayer parallel plane, is a stochastic process that follows a log-normal distribution, compatible with a pore formation mechanism. Furthermore, the vertical individual force-clamp trajectories, occurring in the bilayer orthogonal bilayer plane, reveal that rupturing process occurs through distinct intermediate mechanical transition states that can be ascribed to the fine chemical composition of the hydrated phospholipid moiety. Altogether, these results provide a first description of unanticipated complexity in the energy landscape governing the mechanically induced bilayer rupture process.

Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy.

Proteins respond to mechanical force by undergoing conformational changes and altering the kinetics of their interactions. However, the biophysical relationship between mechanical force and the lifetime of protein complexes is not completely understood. In this chapter, we provide a step-by-step tutorial on characterizing the force-dependent regulation of protein interactions using in vitro and in vivo single-molecule force clamp measurements with an atomic force microscope (AFM). While we focus on the force-induced dissociation of E-cadherins, a critical cell-cell adhesion protein, the approaches described here can be readily adapted to study other protein complexes. We begin this chapter by providing a brief overview of theoretical models that describe force-dependent kinetics of biomolecular interactions. Next, we present step-by-step methods for measuring the response of single receptor-ligand bonds to tensile force in vitro. Finally, we describe methods for quantifying the mechanical response of single protein complexes on the surface of living cells. We describe general protocols for conducting such measurements, including sample preparation, AFM force clamp measurements, and data analysis. We also highlight critical limitations in current technologies and discuss solutions to these challenges.

Force generation by titin folding.

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments, the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles.

Implementation and Tuning of an Optical Tweezers Force-Clamp Feedback System.

Feedback systems can be used to control the value of a system variable. In optical tweezers, active feedback is often implemented to either keep the position or tension applied to a single biomolecule constant. Here, we describe the implementation of the latter: an optical force-clamp setup that can be used to study the motion of processive molecular motors under a constant load. We describe the basics of a software-implemented proportional-integral-derivative (PID) controller, how to tune it, and how to determine its optimal feedback rate. Limitations, possible feed-forward applications, and extensions into two- and three-dimensional optical force clamps are discussed. The feedback is ultimately limited by thermal fluctuations and the compliance of the involved molecules. To investigate a particular mechanical process, understanding the basics and limitations of the feedback system will be helpful for choosing the proper feedback hardware, for optimizing the system parameters, and for the design of the experiment.

Mechanically Watching the ClpXP Proteolytic Machinery.

Energy-dependent protein degradation is studied through the dual bead ClpXP motility assay. Processing of folded proteins involves recognition, unfolding, translocation, and degradation stages. A dual optical trap, in a passive force-clamp geometry, exhibits bead-to-bead displacements that directly follow subprocesses underlying protein degradation. Discrete nanometer-scale displacements of the bead position reveal steps, dwells and pauses during the unfolding and translocation substeps. With a few structural modifications to the protease machinery and an engineered substrate, the assay represents a "chassis" for the measurement of a wide range of substrates and related machinery. The methods described faithfully record our assay as implemented, including substrate design, wet assay preparation, and the motility assay experiment protocol. The strategies herein permit adaptation of the ClpXP mechanical assay to a wide range of protein degradation systems.

Molecular force spectroscopy on cells.

Molecular force spectroscopy has become a powerful tool to study how mechanics regulates biology, especially the mechanical regulation of molecular interactions and its impact on cellular functions. This force-driven methodology has uncovered a wealth of new information of the physical chemistry of molecular bonds for various biological systems. The new concepts, qualitative and quantitative measures describing bond behavior under force, and structural bases underlying these phenomena have substantially advanced our fundamental understanding of the inner workings of biological systems from the nanoscale (molecule) to the microscale (cell), elucidated basic molecular mechanisms of a wide range of important biological processes, and provided opportunities for engineering applications. Here, we review major force spectroscopic assays, conceptual developments of mechanically regulated kinetics of molecular interactions, and their biological relevance. We also present current challenges and highlight future directions.

DNA condensation by TmHU studied by optical tweezers, AFM and molecular dynamics simulations.

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.