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We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component. The soft component visco-elastically changed in a characteristic fashion depending on the physiological conditions of myofibrils, suggesting that it comes from cross-bridge structures. BDM treatments significantly affected the soft radial component of contracting myofibrils depending on the approach velocity of cantilever: It was nearly equal to that in the contracting state at high approach velocity, whereas as low as that in the relaxing state at low approach velocity. However, comparable BDM treatments greatly suppressed the force production and the axial stiffness in contracting glycerinated muscle fibers and also the sliding velocity of actin filaments in the in vitro motility assay. Considering that BDM shifts the cross-bridge population from force generating to pre-force generating states in contracting muscle, the obtained results strongly suggest that cross-bridges in the pre-force generating state are visco-elastically attached to the thin filaments in such a binding manner that the axial stiffness is low but the radial stiffness significantly high similar to that in force generating state.

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The radial stability of the actomyosin filament lattice in skeletal myofibrils was examined by using atomic force microscopy. The diameter and the radial stiffness of the A-band region were examined based on force-distance curves obtained for single myofibrils adsorbed onto cover slips and compressed with the tip of a cantilever and with the Dextran treatment. The results obtained indicated that the A-band is composed of a couple of stiffness components having a rigid core-like component. It was further clarified that these radial components changed the thickness as well as the stiffness depending on the physiological condition of myofibrils. Notably, by decreasing the ionic strength, the diameter of the A-band region became greatly shrunken, but the rigid core-like component thickened, indicating that the electrostatic force distinctly affects the radial structure of actomyosin filament components. The results obtained were analyzed based on the elementary structures of the filament lattice composed of cross-bridges, thin filaments and thick filament backbones. It was clarified that the actomyosin filament lattice is radially deformable greatly and that (1), under mild compression, the filament lattice is stabilized primarily by the interactions of myosin heads with thin filaments and thick filament backbones, and (2), under severe compression, the electrostatic repulsive interactions between thin filaments and thick filament backbones became predominant.

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Caldicellulosiruptor bescii is a cellulolytic/hemicellulolytic anaerobe, which extracellularly secretes various proteins, including multidomain cellulases with two-catalytic domains, for plant biomass degradation. Degradation by C. bescii cells has been well characterized, but degradation by the cell-free extracellular cellulase/hemicellulase system (CEC) of C. bescii has not been as well studied. In the present study, C. bescii CEC was prepared from cell-free culture supernatant, and the degradation properties for defined substrates and non-pretreated plant biomass were characterized. Four multidomain cellulases (Cbes_1857, Cbes_1859, Cbes_1865, and Cbes_1867), composed of the glycoside hydrolase families 5, 9, 10, 44, and 48, were the major enzymes identified in the CEC by mass spectrometry. The CEC degraded xylan, mannose-based substrates, ß-1,4-linked glucans, including microcrystalline cellulose (Avicel), and non-pretreated timothy grass and rice straw. However, degradation of chitin, pectin, dextran, and wheat starch was not observed. The optimum temperatures for degradation activities were 75°C for timothy grass and Avicel, 85°C for carboxylmethyl cellulose, and >85°C for xylan. The optimum pH for these substrates was 5-6. The degradation activities were compared with a CEC derived from the fungus Trichoderma reesei, the most common enzyme used for plant biomass saccharification. The amounts of degraded Avicel, timothy grass, and rice straw by C. bescii CEC were 2.2-2.4-fold larger than those of T. reesei CEC. The high hydrolytic activity of C. bescii CEC might be attributed to the two-catalytic domain architecture of the cellulases.

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Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of ß-lactoglobulin and anti-bovine ß-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between ß-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between ß-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the ß-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the ß-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.

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By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s(-1)). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s(-1)), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.

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We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody-antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.

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Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto-S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto-S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the "conventional" myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.

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The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.

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Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.

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For quantitative analysis of contractile proteins of muscle by means of X-ray diffraction, it is important to know how the intensities of individual reflections are related to the number of diffracting objects, i.e., the amount of constituent contractile protein in the muscle cell. Here we diffused various amounts of exogenous myosin subfragment-1 (S1) into overstretched skinned skeletal muscle fibers, either in the presence or absence of Ca2+ , and derived the relationship between the S1 content and the intensities of reflections arising from the S1. In theory, the intensities should be proportional to the square of the S1 content (square law). However, the intensity-content relation deviated systematically from the square law as the S1 content was lowered, and it was better described as a linear function at the lower end of the S1 contents (<20% of saturation level). Model calculations show that the way of deviation is explained by the cooperative manner of S1 binding to the regulated thin filament.

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Structural changes of contractile proteins were examined by millisecond time-resolved two-dimensional x-ray diffraction recordings during relaxation of skinned skeletal muscle fibers from rigor after caged ATP photolysis. It is known that the initial dissociation of the rigor actomyosin complex is followed by a period of transient active contraction, which is markedly prolonged in the presence of ADP by a mechanism yet to be clarified. Both single-headed (overstretched muscle fibers with exogenous myosin subfragment-1) and two-headed (fibers with full filament overlap) preparations were used. Analyses of various actin-based layer line reflections from both specimens showed the following: 1), The dissociation of the rigor actomyosin complex was fast and only modestly decelerated by ADP and occurred in a single exponential manner without passing through any detectable transitory state. Its ADP sensitivity was greater in the two-headed preparation but fell short of explaining the large ADP effect on the transient active contraction. 2), The decay of the activated state of the thin filament followed the time course of tension more closely in an ADP-dependent manner. These results suggest that the interplay between the reattached active myosin heads and the thin filament is responsible for the prolonged active contraction in the presence of ADP.

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Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLL's) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLL's of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALL's. On close examination of the mouse pattern, however, a few lower order ALL's were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.

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By applying AFM technology, we studied mechanical characteristics of myofibrils of skeletal muscle. The obtained results indicate that (1) the Z-band is the most rigid sarcomere component stabilizing the myofibril structures, (2) various filamentous components are inter-connected in sarcomere with sufficient mechanical strength to support the contractile force, and (3) the molecular structure of the overlap region between actin and myosin filaments is anisotropic. In any case the present studies clearly indicate that the AFM technique is a powerful tool to investigate the mechanical characteristics of sarcomere structure of muscle fiber.

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The motions of myosin filaments actively sliding along suspended actin filaments were studied. By manipulating a double-beam laser tweezers, single actin filaments were suspended between immobilized microbeads. When another beads coated with myosin filaments were dragged to suspended actin filaments, the beads instantly and unidirectionally slid along the actin filaments. The video image analysis showed that the beads slid at a velocity of ca. 3-5 microm/s accompanied with zigzag motions. When beads were densely coated with myosin filaments, the sliding motions became straight and smooth. The obtained results indicate that (1) during the sliding motions, the interaction between myosin heads and actin filaments is weak and susceptible to random thermal agitations, (2) the effects of thermal agitations to the sliding motions of myofilaments are readily suppressed by mechanical constraints imposed to the filaments, and (3) the active sliding force is produced almost in parallel to the filaments axis.

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A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of approximately 2.5-microm-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.

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