Optical Trapping Assay Research Articles

A Comparison of Mechanical Properties of Drosophila and Mouse Myosin 7a

Myosin 7a is an unconventional myosin present in a range of organisms, and is essential in the function of sensory cells. In Drosophila, myosin 7a (D-M7a) is required to maintain bristle structure in Johnston's organ (the auditory center in Drosophila). Equivalently, in mice the absence of myosin 7a (M-M7a) disrupts stereocillia structure which adversely effects vestibular function. D-M7a and M-M7a share good sequence homology. Both have a 5-IQ lever-arm, followed by a single α-helix (SAH) domain, and an SH3 domain separating two MyTH4-FERM domains. Here we use the three-bead optical trap assay to compare the kinetics and mechanics of myosin 7a from insect (Drosophila) and vertebrate (mouse) species. We use a truncated D-M7a construct (D-M7aTD1), cropped after the SH3 domain to prevent auto-regulation. Due to difficulties with M-M7a expression, a shorter construct truncated after the SAH was used (M-M7aSAH). Data were taken at 50mM ionic strength with 10μM ATP. Step sizes of 10 and 18nm were observed for the D-M7aTD1 and M-M7aSAH, respectively. Variations in light chain binding, and geometric hindrances resulting from the shorter tail in M-M7aSAH, may contribute to this difference. The dwell time data from D-M7aTD1 were fitted well by a single exponential, giving an actin detachment rate (Kdet) of 1.1s−1. This compares favourably with the biochemically determined ADP release step. Interpretation of the dwell time data from M-M7aSAH is less straight forward. The data is poorly fit by a single exponential. A double exponential gives fast (9.5s−1) and slow (0.8s−1) Kdet rates. Comparison to biochemical data suggests the fast rate is related to ADP release, whereas the slow rate may represent ATPase cycling. We conclude Drosophila and mouse myosin 7a exhibit generally similar mechanical properties, though appear differently tuned, perhaps for their species specific function.

Actin Crosslinking Proteins Recognize Distinct Arrangements of Actin Filaments

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that comprise higher order structures, such as lamellar meshes, filopodial bundles and stress fibers. Each of these cytoskeletal structures contains actin filaments and crosslinking proteins, but the role of crosslinking proteins in the initial steps of structure formation has not been clearly elucidated. We employ a novel optical trapping assay to investigate the behaviors of fascin and alpha-actinin during the first steps of structure assembly. Here we show that these proteins have distinct binding characteristics that cause them to recognize and crosslink filaments that are arranged with specific geometries. Alpha-actinin is a promiscuous crosslinker, linking filaments over a range of angles. These angles include parallel (00), anti-parallel (1800), and series of intermediate crossing angles (15-1650). It is also an extremely flexible crosslinker, maintaining connection even when the link is rotated. Conversely, fascin is extremely selective, only crosslinking filaments in a parallel orientation. This means that for fascin bundles to form the actin filaments must occupy a parallel orientation before the structure can be stabilized. In the case of alpha-actinin structures, crosslinks could form at any point during the process of structure assembly. We have shown that crosslinking proteins recognize specific orientations of actin filaments, which places constraints on how cytoskeletal structures assemble and organize.

Folding of the Thiamine Pyrophosphate (TPP) Riboswitch

TPP riboswitches regulate the expression of thiamine-synthesis (thi) genes through a variety of mechanisms, all of which involve binding of TPP to a structured aptamer formed in the untranslated region (UTR) of a thi mRNA. We used a high-resolution, single-molecule optical trapping assay to characterize mechanically the folding of the TPP riboswitch aptamer located in the 3’UTR of the thiC gene of Arabidopsis thaliana. Each RNA molecule, containing either the complete aptamer sequence or a portion thereof, was transcribed in vitro, annealed to DNA handles via single-stranded overhangs, and placed in a “dumbbell” experimental geometry1. By applying tension to the ends of the RNA molecule under equilibrium conditions and measuring the corresponding extensions, we observed transitions among several well-defined folding states, which we discuss in the context of secondary and tertiary structures formed by the aptamer2. One low-force state of the full aptamer, corresponding to the formation of structural elements located near the three-helix junction, was abolished by mutating a single nucleotide believed to participate in specific tertiary contacts within the junction2,3. We observed that the mutant aptamer does not bind TPP or other substrates (thiamine, thiamine monophosphate), and that the wild-type aptamer only binds substrates concomitant with entry into the fully-folded state. We also studied the energetics of substrate binding under non-equilibrium conditions by rapidly increasing or decreasing the extension of the aptamer and measuring the hysteresis in force. The number of phosphates on the substrate modulated the amount of work required to induce substrate unbinding, the height and location of the energy barrier to substrate unbinding, and the amount of RNA released.1. Greenleaf WJ, et al (2005). PRL 95, 208102.2. Thore S, et al (2006). Science 312, 1208-1211.3. Sudarsan N, et al (2005). Chemistry & Biology 12, 1325-1335.

Processive Non-Muscle Myosin IIB Takes Load-Independent Backward Steps

Non-muscle myosin IIB (NMIIB) is a molecular motor involved in the regulation of cell polarity during cellular migration. NMIIB forms thick filaments like other members of the myosin II family. Recent studies have shown that a NMIIB dimer can bind both heads to actin simultaneously with different ADP release rates from leading and trailing heads. Gating of ADP release suggests that the two heads communicate with each other and may be capable of processive stepping. We performed single molecule optical trapping assays to examine the stepsize and dwelltime of NMIIB on actin filaments. Our results show that NMIIB is an unconventional myosin that walks processively by taking 5.5 nm backward and forward steps along the long-pitch helix of actin filaments. Forward steps and detachment are weakly force dependent, suggesting ADP release is the rate-limiting step in these transitions. Backward steps are independent of force, suggesting that backward steps occur before ADP release in the lead head. Nucleotide independent backstepping could be a common mechanism for back steps in myosin.

Conformational Dynamics of Single RecBCD Molecules

RecBCD is a multifunctional enzyme possessing both helicase and nuclease activities. It harnesses the energy of ATP hydrolysis to processively unwind DNA. We used an optical-trapping assay featuring one base-pair stability to investigate the mechanism of RecBCD unwinding. Records of RecBCD motion at 6 pN of applied load showed fluctuations [4.1 ± 0.1 bp, (mean ± std. err.; freq. bandwidth = 0.1-10 Hz)] substantially above the control records with DNA alone. These fluctuations persisted when the enzyme's forward motion was stopped by removing ATP. Records of RecBCD bound to blunt-end DNA in the absence of ATP showed reduced dynamics (2.4 ± 0.2 bp), indicating the primary origin of the fluctuations was not due to anchoring via RecBCD. Prior biochemical studies showed that unwinding activity is preceded by an initiation phase consisting of several kinetic steps that generates a 10-nt, 5′-tailed substrate inside the RecBCD-DNA complex that engages RecD's helicase domain. This work also showed that binding to a forked 3′-(dT)6 and 5′-(dT)6 DNA substrate is kinetically equivalent to binding to a blunt-end DNA, while a 3′-(dT)6 and 5′-(dT)10substrate bypasses initiation. We found that records of RecBCD bound to these tailed DNA substrates showed fluctuations that quantitatively mirrored our records of RecBCD bound to blunt-end DNA and stopped within a long DNA substrate, respectively. Thus, the onset of large fluctuations in the RecBCD-DNA complex was coincident with that of unwinding activity. The magnitude and frequency of fluctuations increased when the DNA sequence immediately in front of the forked substrate was changed from GC to AT base pairs, consistent with RecBCD transiently translocating along the DNA without ATP hydrolysis. A tightly bound state with reduced dynamics (2.7 ± 0.1 bp) was observed with ADP-BeF2. These findings support a ratchet model for RecBCD movement.

Parametric Analysis for Monitoring 2D Kinetics of Receptor–Ligand Binding

Thermal fluctuation approach is widely used to monitor association kinetics of surface-bound receptor–ligand interactions. Various protocols such as sliding standard deviation (SD) analysis (SSA) and Page’s test analysis (PTA) have been used to estimate two-dimensional (2D) kinetic rates from the time course of displacement of molecular carrier. In the current work, we compared the estimations from both SSA and modified PTA using measured data from an optical trap assay and simulated data from a random number generator. Our results indicated that both SSA and PTA were reliable in estimating 2D kinetic rates. Parametric analysis also demonstrated that such the estimations were sensitive to parameters such as sampling rate, sliding window size, and threshold. These results furthered the understandings in quantifying the biophysics of receptor–ligand interactions.

Integrating a High-Force Optical Trap with Gold Nanoposts and a Robust Gold−DNA Bond

Gold-thiol chemistry is widely used in nanotechnology but has not been exploited in optical-trapping experiments due to laser-induced ablation of gold. We circumvented this problem by using an array of gold nanoposts (r = 50-250 nm, h approximately 20 nm) that allowed for quantitative optical-trapping assays without direct irradiation of the gold. DNA was covalently attached to the gold via dithiol phosphoramidite (DTPA). By using three DTPAs, the gold-DNA bond was not cleaved in the presence of excess thiolated compounds. This chemical robustness allowed us to reduce nonspecific sticking by passivating the unreacted gold with methoxy-(polyethylene glycol)-thiol. We routinely achieved single beads anchored to the nanoposts by single DNA molecules. We measured DNA's elasticity and its overstretching transition, demonstrating moderate- and high-force optical-trapping assays using gold-thiol chemistry. Force spectroscopy measurements were consistent with the rupture of the strepavidin-biotin bond between the bead and the DNA. This implied that the DNA remained anchored to the surface due to the strong gold-thiol bond. Consistent with this conclusion, we repeatedly reattached the trapped bead to the same individual DNA molecule. Thus, surface conjugation of biomolecules onto an array of gold nanostructures by chemically and mechanically robust bonds provides a unique way to carry out spatially controlled, repeatable measurements of single molecules.

Single molecule transcription elongation

Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.

Precision Surface-Coupled Optical-Trapping Assay with One-Basepair Resolution

The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale (∼0.1 nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (<0.05 nm) of bead detection over a broad range of trap stiffness ( k T = 0.05–0.5 pN/nm) and frequency (Δ f = 0.03–100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision (∼0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.

Non-muscle Myosin IIb Is A Processive Actin-based Motor

Proper tension maintainence in the cytoskeleton is essential for regulated cell polarity, cell motility and division. Non-muscle myosin IIB (NmIIB) generates tension in the actin cortex of non-muscle cells. Recent biochemical studies show that both heads of a NmIIB dimer can interact with a single actin filament and that this conformation demonstrates load dependent release of ADP. Using a three bead optical trapping assay we recorded NmIIB interactions with actin filaments to determine if a NmIIB dimer cycles along an actin filament in a processive manner. Our results show for the first time that NmIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of this data reveals a forward displacement of ∼5 nm. Surprisingly, NmIIB can and does take frequent backward steps of ∼5 nm. The short step size of NmIIB suggests that this motor twists actin. Actin twisting could facilitate the removal of actin crosslinking proteins from the cytoskeleton. Our data supports a model in which NmIIB takes processive forward steps to generate additional tension and also takes backwards steps to relieve tension in the actin cytoskeleton, suggesting that NmIIb is a general regulator of cytoskeleton tension.

Human TBP Bending of DNA Measured at the Single Molecule Level

TATA binding protein (TBP) binds to DNA and bends it as a critical step of eukaryotic transcription. Human TBP induces an ∼100° bend in the DNA helix. To help elucidate the role of DNA architecture in transcription, we developed a single-molecule, optical-trapping assay to study the TBP-induced bending of DNA. We hypothesized that DNA bending would lead to an apparent shortening of the DNA. The predicted signal size is small (∼5 nm at 1 pN) and expected to decrease with increasing force. Moreover, the dynamics of TBP are slow, with a measured off-rate of ∼10−2 s−1 at physiological salt conditions. To detect these small, infrequent events, we developed an actively stabilized optical trapping instrument. We achieved high spatiotemporal resolution [0.56 nm (Δf = 0.01-1 Hz) over 200 s] at low force (1 pN) by developing a vertical optical trapping assay using short DNA tethers (92 nm) along with small beads (330 nm dia.). Detection of distinct TBP and TATA-box dependent signals required DNA molecules engineered to contain only one consensus TATA-box with no TATA-box like sequences in the flanking DNA. Significant nonspecific binding to the glass surfaces was minimized by covalently attaching polyethylene glycol (PEG) to the glass. With these experimental procedures, we directly observed individual bending and unbending events. The TBP-dependent DNA conformational changes were dynamic on the timescale of tens of seconds at a [TBP] ≈ 20 nM and a force of 1 pN. The change in DNA extension (∼5 nm) agreed well with theoretical predictions at 1 pN, but, unlike the simplest theory, showed little change in magnitude as the force was decreased. We expect that this assay will be broadly useful for studying DNA-protein interactions at high spatiotemporal resolution.

Sensitivity Of Dna-hairpins Dynamics To The Mechanism Of Force Feedback Probed Using A Surface-coupled Passive Force Clamp

Optical-trapping experiments have yielded new insight into the mechanical behavior of individual biomolecules. A common experimental assay consists of an enzyme or nucleic acid molecule attached to a cover slip at one end and to a small polystyrene bead at the other. The bead is captured and held under tension with an optical trap. Active feedback maintains constant force, often called a force clamp, to increase measurement precision. Yet, active feedback is inherently bandwidth limited. This limited bandwidth leads to significant fluctuations in force that are particularly pronounced for rapid, large changes in molecule extension (e.g. DNA hairpin unfolding). A novel, passive force clamp circumvents this limitation by pulling the bead to a non-linear region of the trap where ktrap = 0. To date, this passive force clamp has required a specialized dual optical trapping apparatus where one trap measures position and the other measures force. Here, we demonstrate a passive force clamp achieved with a single trap in a surface-coupled assay using a previously characterized DNA hairpin. In an active force clamp, rapid back-and-forth transition between open and closed hairpins states were observed within the update period of the active force clamp (2 or 10 ms) as well as a change in the long term dynamics. By using a passive force clamp, these spurious transitions were eliminated and the correct dynamics measured. By analyzing the fluctuations in the bead position in conjunction with the known elasticity of DNA, we simultaneously measured force and position in a single-beam, passive force clamp. Thus, the benefits of the passive force clamp are now available to the widely used surface-coupled optical trapping assays.

Direct Observation of Individual Kinesin Head Motions

Optical-trapping assays for kinesin typically involve attaching a bead to the common stalk of the protein, whose motions report the average position of the molecule. Individual displacements produced by the separate heads therefore remain unresolved. We developed a novel assay for tracking a single head of Kinesin-1 while under controlled loads, by attaching a bead to one of the two head domains via a short (70 bp) DNA tether. This assay can directly report binding of the tethered head to the microtubule. Under hindering loads, we observed steps of ∼16 nm, as anticipated for heads moving in a hand-over-hand walk. Under assisting loads, we observed large jumps (>16-nm) in displacement at the start of step dwells, as load pulled the rear head forward beyond its partner head by ∼4 nm. Torque generated between the points of head attachment of the DNA and the neck linker tends to rotate the head, explaining this ‘overshoot’ feature. The durations of overshoots depend on the ATP concentration, implying that ATP binding to the new rear head allows the front (DNA-linked) head to rotate back to its normal orientation and bind the microtubule. To directly test whether one head is free to diffuse about its bound partner head between steps, we applied rapidly oscillating (hindering and assisting) loads to kinesin during stepping and measured the time-dependent difference between forward and rearward displacements of the bead. The magnitude of this signal varied between two discrete values, corresponding to those intervals when the bead-linked head adopted bound and unbound states. The existence of an unbound state disfavors models where one head docks against its partner between steps. We conclude that internal strain, generated whenever both heads bind the microtubule, is responsible for gating the kinetic cycle to ensure kinesin processivity.

Modulation Of Sequence-dependent Pausing Of RNA Polymerase By The Accessory Factor Nusa: A Single-molecule Study

Sequence-dependent pausing during transcription by RNA polymerase (RNAP) plays an essential role in the regulation of gene expression and is modified by timely interactions with a number of regulatory factors. In prokaryotes, NusA is a universally conserved accessory protein that is essential for cell viability and functions to modulate sequence-dependent pausing. Although NusA is known to both decrease the average rate of elongation and enhance certain types of pauses (e.g., the RNA hairpin-associated his pause), the kinetic details and mechanism underlying these effects are not well understood. We employed a dumbbell optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual molecules of RNAP transcribing a DNA template engineered to contain repeats of a sequence motif carrying the his pause. From individual transcriptional elongation records, the rates of entering pause states, the pause state lifetimes, and the pause-free elongation speeds can all be extracted. We found that the addition of NusA to the transcriptional assay significantly decreased the pause-free elongation speed while concurrently increasing the efficiency of entry into both long- and short-lifetime pauses. Studying these effects as a function of the applied load led to a quantitative kinetic scheme for elongation and pausing. In this model, the binding of NusA to RNAP functions equivalently to exerting a hindering load of ∼19 pN on RNAP with respect to the DNA. We therefore speculate that the mechanism of NusA may be to modulate RNAP elongation in a mechanochemical sense.

Low Spring Constant Regulates P-Selectin-PSGL-1 Bond Rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r f (≥10 2 pN/s), defined as the product of spring constant k and retract velocity v, how the low r f (<10 2 pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r f ≤ 20 pN/s with low k (∼10 −3–10 −2 pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 × 10 −3 pN/nm, indicating that the bond dissociation at low r f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r f or k.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Direct Observation of Hierarchical Folding in Single Riboswitch Aptamers

Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.

Elasticity of Short DNA Molecules: Theory and Experiment for Contour Lengths of 0.6–7 μm

The wormlike chain (WLC) model currently provides the best description of double-stranded DNA elasticity for micron-sized molecules. This theory requires two intrinsic material parameters—the contour length L and the persistence length p. We measured and then analyzed the elasticity of double-stranded DNA as a function of L (632 nm–7.03 μm) using the classic solution to the WLC model. When the elasticity data were analyzed using this solution, the resulting fitted value for the persistence length p wlc depended on L; even for moderately long DNA molecules ( L = 1300 nm), this apparent persistence length was 10% smaller than its limiting value for long DNA. Because p is a material parameter, and cannot depend on length, we sought a new solution to the WLC model, which we call the “finite wormlike chain (FWLC),” to account for effects not considered in the classic solution. Specifically we accounted for the finite chain length, the chain-end boundary conditions, and the bead rotational fluctuations inherent in optical trapping assays where beads are used to apply the force. After incorporating these corrections, we used our FWLC solution to generate force-extension curves, and then fit those curves with the classic WLC solution, as done in the standard experimental analysis. These results qualitatively reproduced the apparent dependence of p wlc on L seen in experimental data when analyzed with the classic WLC solution. Directly fitting experimental data to the FWLC solution reduces the apparent dependence of p fwlc on L by a factor of 3. Thus, the FWLC solution provides a significantly improved theoretical framework in which to analyze single-molecule experiments over a broad range of experimentally accessible DNA lengths, including both short (a few hundred nanometers in contour length) and very long (microns in contour length) molecules.

Gold nanoparticles: enhanced optical trapping and sensitivity coupled with significant heating

Gold nanoparticles appear to be superior handles in optical trapping assays. We demonstrate that relatively large gold particles (R(b)=50 nm) indeed yield a sixfold enhancement in trapping efficiency and detection sensitivity as compared to similar-sized polystyrene particles. However, optical absorption by gold at the most common trapping wavelength (1064 nm) induces dramatic heating (266 degrees C/W). We determined this heating by comparing trap stiffness from three different methods in conjunction with detailed modeling. Due to this heating, gold nanoparticles are not useful for temperature-sensitive optical-trapping experiments, but may serve as local molecular heaters. Also, such particles, with their increased detection sensitivity, make excellent probes for certain zero-force biophysical assays.

Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules

Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.