Cross-bridges In Muscle Fibers Research Articles

Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

BackgroundPhotoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.Methods and FindingsFluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ2 minimization.ConclusionsResults indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation.

In Situ Fluorescent Protein Imaging with Metal Film-Enhanced Total Internal Reflection Microscopy

Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of ∼120 μM, implying single myosin detection volume for this biological assembly is ∼1 attoL (10 −18 L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from ∼3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM’s usefulness for single molecule isolation within biological assemblies.

Conformation of myosin interdomain interactions during contraction: deductions from proteins in solution.

Myosin subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). These domains interface in two places identified as interface I, containing the reactive thiol (SH1) and ATP-sensitive tryptophan (Trp510), and interface II, containing the reactive lysine residue (RLR). Two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from extrinsic probes located in the interfaces. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Structures giving the best agreement between the computed and observed signals were selected as the representative forms. Both interfaces undergo dramatic conformational change during ATPase and force development. Changes at interface I suggest the molecular basis for the collisional quenching sensitivity of Trp510 to nucleotide binding. The probe conformation at SH1 suggests how it alters S1 ATPases. At interface II, the spatial relationship of the lever arm and the extrinsic probe at RLR suggests how the probe alters S1 ATPases and that it should inhibit lever arm movement during the power stroke. The latter possibility, if true, establishes a part of the corridor through which the lever arm swings during the power stroke. Global structural changes in actomyosin are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4821-4833].

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence.

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].

The power stroke causes changes in the orientation and mobility of the termini of essential light chain 1 of myosin.

Binding of ATP to the catalytic domain of myosin induces a local conformational change which is believed to cause a major rotation of an 8.5 nm alpha-helix that is stabilized by the regulatory and essential light chains. Here we attempt to follow this rotation by measuring the mobility and orientation of a fluorescent probe attached near the C- or N-terminus of essential light chain 1 (LC1). Cysteine 178 of wild-type LC1, or Cys engineered near the N-terminus of mutant LC1, was labeled with tetramethylrhodamine and exchanged into skeletal subfragment-1 (S1) or into striated muscle fibers. In the absence of ATP, the fluorescence anisotropy (r) and the rotational correlation time (rho) of S1 reconstituted with LC1 labeled near the C-terminus were 0.195 and 66.6 ns, respectively. In the presence of ATP, r and rho increased to 0.233 and 233 ns, indicating considerable immobilization of the probe. A related parameter indicating the degree of order of cross-bridges in muscle fibers, Deltar, was small in rigor fibers (-0.009) and increased in relaxed fibers (0.030). For S1 reconstituted with LC1 labeled near the N-terminus, the steady-state anisotropy was 0.168 in rigor, and increased to 0.223 in relaxed state. In fibers, the difference in rigor was large (Deltar = 0.080), because of binding to the thin filaments, and decreased to 0.037 in relaxed fibers. These results suggest that before the power stroke, in the presence of ATP or its products of hydrolysis, the termini of LC1 are immobilized and ordered, and after the stroke, they become more mobile and partially disordered. The results are consistent with crystallographic structures that show that the level of putative stabilizing interactions of LC1 with the heavy chain of S1 in the transition state is reduced as the regulatory domain rotates to its post-power stroke position.

Rectified Brownian movement in molecular and cell biology

A unified model is presented for rectified Brownian movement as the mechanism for a variety of putatively chemomechanical energy conversions in molecular and cell biology. The model is established by a detailed analysis of ubiquinone transport in electron transport chains and of allosteric conformation changes in proteins. It is applied to P-type ATPase ion transporters and to a variety of rotary arm enzyme complexes. It provides a basis for the dynamics of actin-myosin cross-bridges in muscle fibers. In this model, metabolic free energy does no work directly, but instead biases boundary conditions for thermal diffusion. All work is done by thermal energy, which is harnessed at the expense of metabolic free energy through the establishment of the asymmetric boundary conditions. @S1063-651X~98!13502-X#

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres.

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S1 during the transition.

Application of angle-resolved fluorescence depolarization in muscle research

Angle-resolved fluorescence depolarization (AFD) experiments have been used for over a decade in studies of fluorescent molecules in macroscopically aligned systems such as lipid bilayers and stretched polymer films. The importance of this technique lies in the fact that it affords the determination of both the second- and the fourth-rank order parameters of the orientational distribution of the probe molecules in the sample. Here we apply the technique to the study of the orientational distribution of crossbridges in muscle fibers. This orientational distribution is particularly relevant in muscle research, as crossbridge rotation is commonly regarded to be the driving mechanism in force development. An unfortunate consequence of the fact that the crossbridges have an average orientation of approximately 45(o) relative to the fiber axis is that the values of the second-rank order parameter [Symbol: see text]P 2[Symbol: see text] of the crossbridge distribution are close to 0. Therefore, knowledge of [Symbol: see text]P 4[Symbol: see text] is essential for a reliable reconstruction of the form of the distribution function. AFD of dyelabeled muscle was measured under rigor and relaxation conditions. The results indicate that no significant changes in depolarization take place upon a transition from the rigor to the relaxed state in the muscle and seem not to support the rotating crossbridge model, which postulates a clear change of orientation of the crossbridges.

Following the rotational trajectory of the principal hydrodynamic frame of a protein using multiple probes.

A generalized set of fluorescence polarization intensities and electron paramagnetic resonance (EPR) spectra from multiple fluorescent and EPR probes of a specific site on an oriented and immobilized protein element in a biological assembly, combined with anisotropic rotational relaxation studies of the purified and labeled protein element freely rotationally diffusing in solution, are used to determine the angular distribution of the principal hydrodynamic frame of the protein element in the biological assembly. This multiprobe analysis method removes all of the basic ambiguities in the measured principal frame angular distribution introduced by limitations or symmetries intrinsic to standard fluorescence polarization intensity ratios and EPR spectra. The angular distribution of the principal frame is also more highly resolved than that previously reported for multiprobe determinations of probe angular distributions and is more useful for determining biological mechanisms because it indicates the order and orientation of the protein element rather than that of the extrinsic probe. Application of this method to the determination of the principal hydrodynamic frame distribution of myosin cross-bridges in muscle fibers in four physiological states, including the active isometric state, demonstrates the method's practicality by indicating the path of cross-bridge orientation changes during the active cycle [Ajtai, Toft, & Burghardt (1994) Biochemistry (following paper in this issue)].

Path and Extent of Cross-Bridge Rotation during Muscle Contraction

The angular distribution of myosin cross-bridges in muscle fibers was investigated in four physiological states using a multiple probe analysis of varied extrinsic probes of the cross-bridge [Burghardt & Ajtai (1994) Biochemistry (preceding paper in this issue)]. The analysis combines data of complementary techniques from different probes giving the highest possible angular resolution. Four extrinsic probes of the fast reactive sulfhydryl (SH1) on myosin subfragment 1 (S1) were employed. Electron paramagnetic resonance (EPR) spectra from paramagnetic probes, deuterium- and 15N-substituted for greater sensitivity to orientation, on S1 were measured when the protein was freely tumbling in solution and when it was decorating muscle fibers. The EPR spectra from labeled S1 tumbling in solution were measured at X- and Q-band microwave frequencies to uniquely specify the orientation of the probe relative to the S1 principal hydrodynamic frame. The EPR spectra from labeled S1 decorating muscle fibers in rigor and in the presence of MgADP were measured at X-band and used in the multiple probe analysis of cross-bridge orientation. The time-resolved fluorescence anisotropy decay (TRFAD) of fluorescent probes on S1 was measured when the protein was freely tumbling in solution, and fluorescence polarization (FP) intensities from fluorescent probes modifying SH1 in intact muscle fibers were measured for fibers in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. The TRFAD measurements limit the range of possible orientations of the probe relative to the S1 principal hydrodynamic frame. The FP intensity measurements were used in the multiple probe analysis of cross-bridge orientation. The combination of the EPR and FP data determined a highly resolved cross-bridge angular distribution in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. These findings confirm earlier observations of a rigid body rotation of the SH1 region in the myosin head group upon physiological state changes and indicate the path and extent of cross-bridge rotation during contraction. The rotation of the cross-bridge is visualized with computer-generated space-filling models of actomysin in six states of the contraction cycle.

The orientation of transition moments of dye molecules used in fluorescence studies of muscle systems.

Fluorescence and phosphorescence depolarization techniques can provide information on orientational order and rotational motion of crossbridges in muscle fibres. However the depolarization experiment monitors the orientation and motion of the crossbridges indirectly. The changes in depolarization arise from a change in the orientation of the transition dipoles of the dye attached to the crossbridge. In order to extract the physiologically relevant orientations from the data it is therefore necessary to characterize the orientation of the dye molecule relative to the crossbridge and the orientation of the transition moments in the frame of the dyes. The dyes 1,5-I-AEDANS and eosin-5-maleimide are commonly used for labelling the crossbridge in muscle fibres. The orientations of the absorption and fluorescence emission dipoles of these two dyes in the molecular frame were determined. Angle resolved fluorescence depolarization experiments on the dyes, macroscopically aligned in a stretched polymer matrix of poly vinyl alcohol, were carried out. The data were analyzed in terms of an orientational distribution of the dye molecules in the film and the orientations of the absorption and emission dipoles in the frame of the dye molecule. Experimental data, obtained from a given sample at different excitation wavelengths, were analyzed simultaneously in a global target approach. This leads to a reduction in the number of independent parameters optimized by the non-linear least squares procedure.

Effect of negative mechanical stress on the orientation of myosin cross-bridges in muscle fibers.

The effect of positive and negative stress on myosin cross-bridge orientation in glycerinated muscle fibers was investigated by using fluorescence polarization spectroscopy of the emission from the covalent label tetramethyl-rhodamine-5-(and -6)-iodoacetamide (IATR) specifically modifying sulfhydryl one (SH1) on the myosin heavy chain. Positive tension was applied by stretching the fiber in rigor. Negative tension was applied in two steps by using a protocol introduced by Goldman et al. [Goldman, Y. E., McCray, J. A. & Vallette, D. P. (1988) J. Physiol. (London) 398, 75P]: relaxing a fiber at resting length and stretching it until the relaxed tension is appreciable and then placing the fiber in rigor and releasing the tension onto the rigor cross-bridges. We found, as have others, that positive tension has no effect on the fluorescence polarization spectrum from the SH1-bound probe, indicating that the cross-bridge does not rotate under these conditions. Negative tension, however, causes a change in the fluorescence polarization spectrum that indicates a probe rotation. The changes in the polarization spectrum from negative stress are partially reversed by the subsequent application of positive stress. It appears that negative tension strains the cross-bridge, or the cross-bridge domain containing SH1, and causes it to rotate.

Model-independent time-resolved fluorescence depolarization from ordered biological assemblies applied to restricted motion of myosin cross-bridges in muscle fibers.

We formulate a model-independent description of time-dependent restricted molecular rotational motion and apply it to the time-resolved fluorescence depolarization signal from fluorescence-labeled molecules following polarized excitation. In this treatment, the time-dependent molecular orientation distribution is equal to the operation of a linear time-development operator on the initial orientation distribution. The formula for the time-development operator is derived from the equation of motion for the orientation distribution. The time-development operator is then expanded in time in terms of a complete set of orthonormal polynomials. When this expression for the orientation distribution is used to calculate the time-resolved fluorescence depolarization signal, properties of the orthonormal polynomials allow the signal to be quantitated uniquely in terms of matrix elements of the differential operators from the equation of motion. We demonstrate how to obtain the experimental value of the matrix elements directly from the fluorescence depolarization signal. In this paper, we also describe the application of the model-independent formalism to fluorescence-labeled myosin cross-bridges in muscle fibers when the fibers are in a variety of physiological states. We show that the analysis of time-resolved fluorescence depolarization data with the new formalism and the rotational diffusion in a potential model results in a slight revision of our previous estimate of the relaxed cross-bridge rotational diffusion constant and the determination of rank 6 order parameters that were ignored in the previous analysis [Burghardt, T. P., & Thompson, N. L. (1985) Biochemistry 24, 3731-3735]. The rank 6 order parameters are shown to make a significant contribution to the proposed steady-state angular distribution of the relaxed cross-bridges. Order parameters of rank 6 do not contribute to the steady-state fluorescence polarization signal and can only be detected with the time-resolved signal [Burghardt, T. P. (1984) Biopolymers 23, 2383-2406]. We have also applied the model-independent formalism to new data from fibers in rigor when the fibers are in a configuration such that the excitation light polarization is perpendicular to the fiber axis and the light propagates along the fiber axis, so that the fluorescence depolarization signal is sensitive to probe rotational motions about the fiber axis.(ABSTRACT TRUNCATED AT 400 WORDS)

Model-independent electron spin resonance for measuring order of immobile components in a biological assembly

A model-independent description of the angular orientation distribution of elements in an ordered biological assembly is applied to the electron spin resonance (ESR) technique. As in a previous model-independent treatment of fluorescence polarization (Burghardt, T.P., 1984, Biopolymers, 23:2383-2406) the elemental order is described by an angular distribution of molecular frames with one frame fixed in each element of the assembly. The distribution is expanded in a complete orthonormal set of functions. The coefficients of the series expansion (the order parameters) describe the orientation distribution of the elements in the assembly without reference to a model and can be obtained from the observed spectrum. The method establishes the limitations of ESR in detecting order in the assembly by determining which distribution coefficients the technique can detect. A method of determining the order parameters from an ESR spectra, using a set of ESR basis spectra, is developed. We also describe a treatment that incorporates the actual line shape measured from randomly oriented, immobile elements. In this treatment, no model-dependent assumptions about the line shape are required. We have applied the model-independent analysis to ESR spectra from spin-labeled myosin cross-bridges in muscle fibers. The results contain detailed information on the spin-probe angular distribution and differ in interesting ways from previous model-dependent interpretations of the spectra.

Three-dimensional disorder of dipolar probes in a helical array. Application to muscle cross-bridges

Fluorescence polarization and EPR experiments on azimuthally randomized helices bearing extrinsic (dipolar) probes yield information about the axial orientation and order of the probes. If the orientation of the probe on the structure bearing it is known and disorder is absent, the orientation of the structure may be ascertained. For cases where less probe orientation information is available and/or disorder is present, the available structural information is correspondingly reduced. Here we examine the available data on probes attached to cross-bridges in muscle fibers: four plausible cases of three-dimensional cross-bridge disorders are numerically modeled muscle in states of rigor and relaxation. In rigor, where the reported probe disorder is small (Thomas and Cooke, 1980), it was found that the cross-bridge disorder was also small. On the other hand, for the relaxed state where the probes are found to be completely disordered, the cross-bridges may have a considerable amount of order. This possibility is in concert with the results of x-ray diffraction, in which the presence of well-developed myosin-based layer lines indicates considerable order in relaxed muscle.