Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy.

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

Data on whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

Data presented in this article relates to the research article entitled "Whole length myosin binding protein C stabilizes myosin subfragment-2 (S2) flexibility as measured by gravitational force spectroscopy." (Singh et al., 2018) [1]. The data exhibits the purified skeletal myosin binding protein C (MyBPC) from rabbit back muscle was of slow skeletal type confirmed by chromatography and in unphosphorylated state based on its isoelectric point (pI) by chromatofocussing. The competitive enzyme linked immunosorbent assay (cELISA) data displayed the site specificity of polyclonal anti-S2 antibody to myosin S2. This polyclonal antibody binding site corresponds to a familial hypertrophic cardiomyopathy (FHC) point mutation hotspot on myosin S2 illustrated in a figure of compiled data.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem.

The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA.

Asymmetric myosin binding to the thin filament as revealed by a fluorescent nanocircuit.

The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the pre powerstroke orientation when bound near troponin, but is predominantly in the post powerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament.

Demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence. The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

Close proximity of myosin loop 3 to troponin determined by triangulation of resonance energy transfer distance measurements.

Cooperative activation of the thin filament is known to be influenced by the tight binding of myosin to actin, but the molecular mechanism underlying this contribution of myosin is not well understood. To better understand the structural relationship of myosin with the regulatory troponin complex, resonance energy transfer measurements were used to map the location of troponin relative to a neighboring myosin bound to actin using atomic models. Using a chicken troponin T isoform that contains a single cysteine near the binding interface between troponins T, I, and C, this uniquely labeled cysteine on troponin was found to be remarkably near loop 3 of myosin. This loop has previously been localized near the actin and myosin interface by chemical cross-linking methods, but its functional contributions have not been established. The implications of this close proximity are examined by molecular modeling, which suggests that only restricted conformations of actomyosin can accommodate the presence of troponin at this location near the cross-bridge. This potential for interaction between troponin and myosin heads that bind near it along the thin filament raises the possibility of models in which direct myosin and troponin interactions may play a role in the regulatory mechanism.

Fluorescence labeling and computational analysis of the strut of myosin's 50kDa cleft.

A new fluorescent labeling procedure specific for the strut sequence of myosin subfragment-1's 50kDa cleft was developed using CY3 N-hydroxy succinimidyl ester as a hydrophobic tag and hydrophobic interaction chromatography to purify the major labeled species which retained actin-activated ATPase activity. Stern-Volmer analysis suggests that the CY3 is in close proximity to basic residues, consistent with inspection of the mapped labeling site in the atomic model. Fluorescence polarization indicates that the CY3 becomes more mobile upon actin binding, supporting a location near the actomyosin interface. In contrast, nucleotide binding to myosin had little impact on the CY3. Molecular mechanics and stochastic dynamics simulations suggest that this labeling site is sensitive to forced cleft opening and closure, but the upper 50kDa cleft does not move easily. In addition, there appear to be some long-range effects of forced cleft opening and closing that could impact the lever arm position.

Downscaling functional bioassays by single-molecule techniques.

In this short review we examine the potential of single-molecule assays in drug development and in basic research to provide new types of information at the smallest assay scales. A key advantage of many single-molecule assays is the requirement for conservative amounts of precious sample compared to conventional assays. In addition, they measure processes that are not observed directly in molecular ensembles. These advantages are balanced currently by difficulties in assay setup, preparation and equipment expense. However, future developments will ameliorate these drawbacks with the production of simpler, less expensive experimental systems for single-molecule assays.

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin.

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.

High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay.

Popular views of force generation in muscle indicate that a lever arm in the myosin head initiates displacement of the thin filament. However, this lever arm is attached to the thick filament backbone by a flexible combination of coiled coils and hinges in the myosin subfragment-2 (S2); therefore, efficient force generation depends on tension development in this linking structure. Herein, a single molecule assay is developed to examine the flexibility of the intact S2 relative to that of the myosin head. Fluorescently labeled myosin rod is polymerized onto a single myosin molecule that is bound to actin, and the resulting Brownian motion of the rod is analyzed at video rates by digital image processing. Complete rotations of the rod suggest significant amounts of random coil in the linking structure. The close similarity of twist rates for double-headed and single-headed myosin indicates that most of the flexibility originates at or beyond the first pitch of coiled coil in S2 and most likely at the hinge connecting S2 and the light meromyosin. The myosin head has a smaller but still detectable impact on this flexibility, since the addition of ADP to the rigor crossbridge produces differential effects on the torsional characteristics of double-headed versus single-headed myosin.

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer.

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

Binding of calcium ions to an avian flight muscle troponin T.

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its N-terminal hypervariable region, but the functions of these isoforms are not completely understood. Here for the first time, we discovered that a chicken fast TnT isoform with a unique Tx motif (HEEAH)(n) binds calcium. The metal binding behavior of this TnT isoform was first investigated using terbium as a calcium analogue due to its more readily detectable fluorescence variation upon TnT binding. Both intact TnT and TnT N-terminal fragment (TnT N47) bound terbium with high affinity indicating that the N-terminal sequence was the site of binding. Since terbium often substitutes at calcium-binding sites, radioactive calcium was tested and found to bind both intact TnT and TnT N47. Fluorescence measurements using the calcium-sensitive fluorescent dye, calcium green 5N, confirmed that calcium bound to the tertiary complex of TnT and the tropomyosin dimer with a fast on-rate (10(6)-10(7) M(-1) s(-1)) as detected in stopped-flow analysis. Consistent with these observations, computational predictions suggest that TnT N47 might fold into an elongated structure with at least one high-affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. These results suggest that TnT may play a role in modulating the calcium-mediated regulatory process of striated muscle contraction.

A computational comparison of the atomic models of the actomyosin interface.

Several atomic models of the actomyosin interface have been proposed based on the docking together of their component structures using electron microscopy and resonance energy-transfer measurements. Although these models are in approximate agreement in the location of the binding interfaces when myosin is tightly bound to actin, their relationships to molecular docking simulations based on computational free-energy calculations are investigated here. Both rigid-docking and flexible-docking conformational search strategies were used to identify free-energy minima at the interfaces between atomic models of myosin and actin. These results suggest that the docking model produced by resonance energy-transfer data is closer to a free-energy minimum at the interface than are the available atomic models based on electron microscopy. The conformational searches were performed using both scallop and chicken skeletal muscle myosins and identified similarly oriented actin-binding interfaces that serve to validate that these models are at the global minimum. These results indicate that the existing docking models are close to but not precisely at the lowest-energy initial contact site for strong binding between myosin and actin that should represent an initial contact between the two proteins; therefore, conformational changes are likely to be important during the transition to a strongly bound complex.

The dance of actin and myosin: a structural and spectroscopic perspective.

Actin and myosin interact in a cyclic series of steps linked to the hydrolysis of ATP that are representative of an ancient and widespread molecular mechanism. Spectroscopic findings are related to the analysis of the actin and myosin structures and results from kinetics, fibers, single molecules, electron microscopy, genetics, and a variety of other biophysical and biochemical studies on actin and myosin to provide an overview of the steps in this molecular process. The synthesis of the key findings from these fields reveals a highly efficient engine that amplifies subtle changes in the active site into unsurpassed molecular displacements. Recent developments in resonance energy-transfer spectroscopy and X-ray crystallography are enabling a detailed elucidation of the stages of a large power stroke that concurs with evidences from diverse lines of structural and kinetic inquiry. A complete image of actin and myosin motility appears to include twists, tilts, steps, and dynamics from both partners that could be described as a molecular dance.

Dynamic docking of myosin and actin observed with resonance energy transfer.

Atomic models of myosin subfragment-1 (S1) and the actin filament are docked together using resonance energy-transfer data from both pre- and postpowerstroke conditions. The quality of the resulting best fits discriminated between neck-region orientations of the S1 for a given set of experimental conditions. For measurements of the postpowerstroke states in the presence of ADP, resonance energy-transfer data alone are sufficient to dock the atomic models and provide evidence that S1 exists with at least two neck-region orientations under these conditions. To dock the prepowerstroke state, resonance energy-transfer data were used in combination with previous chemical cross-linking data to determine that a neck-region orientation similar to that of a proposed prepowerstroke state best fit the data. The resulting models determined independently from electron microscopy compare favorably with micrographs from the recent literature. The docking models by resonance energy transfer suggest that the larger movements in the light-chain binding domain are accompanied by twisting and rotating movements of the catalytic domain, causing a tilt of approximately 30 degrees during the weak-to-strong transition. This transition provides the displacement necessary to support motility and force generation.