Translate this page to:
In JoVE (1)
- Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins
Other Publications (10)
Articles by Douglas D. Root in JoVE
Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins
James W. Dunn, Douglas D. Root
Department of Biological Sciences, University of North Texas
This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.
Other articles by Douglas D. Root on PubMed
Biochemistry. Feb, 2002 | Pubmed ID: 11827523
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.
Cell Biochemistry and Biophysics. 2002 | Pubmed ID: 12482134
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.
Cell Biochemistry and Biophysics. 2002 | Pubmed ID: 12482135
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.
Biochemistry. Mar, 2004 | Pubmed ID: 14992602
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.
Detection of Single Nucleotide Variations by a Hybridization Proximity Assay Based on Molecular Beacons and Luminescence Resonance Energy Transfer
Biopolymers. Sep, 2004 | Pubmed ID: 15307198
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.
High Flexibility of the Actomyosin Crossbridge Resides in Skeletal Muscle Myosin Subfragment-2 As Demonstrated by a New Single Molecule Assay
Journal of Structural Biology. Feb, 2005 | Pubmed ID: 15681228
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.
Biophysical Journal. Apr, 2006 | Pubmed ID: 16439474
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.
Drug Discovery Today. Jul, 2006 | Pubmed ID: 16793533
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.
Archives of Biochemistry and Biophysics. Dec, 2006 | Pubmed ID: 16949551
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.
Close Proximity of Myosin Loop 3 to Troponin Determined by Triangulation of Resonance Energy Transfer Distance Measurements
Biochemistry. Jan, 2009 | Pubmed ID: 19108638
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.