Recent X-ray diffraction studies of muscle contraction and their implications

Recent studies on the interference fringes in the myosin meridional reflections provide a new source of structural information on cross-bridge movement during mechanical transients and steady shortening. Many observations can be interpreted satisfactorily by the tilting lever-arm model, with some assumptions, including the presence of fixed repeating structures contributing to the M3 and higher-order meridional reflections. In isometric contraction, the lever arms are oriented near the start of the working stroke, with a dispersion of ca+/-20-25 degrees . Upon a rapid release of 10-12 nm, they move to the end of the stroke, with a well-known T2 delay of 1-2 ms. This delay must represent additional processes, which have to occur even in tension-generating heads, or activation of attached heads, which initially do not develop force. Surprisingly, in muscles shortening at moderate loads (0.5-0.6 P0), the mean position of the heads moves only 2-3 nm closer to the M-line than in the isometric case, reminiscent of the Piazzesi-Lombardi model.     

X-ray diffraction studies on cation-collapsed DNA

The polyamines spermidine, spermine and putrescine are now known to induce tertiary collapse of DNA. In this collapsed state DNA assumes a compact toroidal conformation. However, the structural details of DNA in these compact particles and the forces that stabilize the collapsed state are not clear. We show here that the structural arrangement of DNA in this tertiary conformation is determined by the chemical structure of the agent used to collapse. We have used aliphatic triamines (NH₃⁺-(CH₂)₃-NH₂⁺-(CH₂)_n-NH₃⁺ with n = 3, 4, 5 and 8) and diamines (NH₃⁺-(CH₂)_x-NH₃⁺ with x = 2, 3, 4 and 6) to collapse DNA. We find that the Bragg spacing and the calculated interhelical spacing for a hexagonal packing model vary systematically with the length of the methylene bridge. We also find that the ionic strength of the solution has no effect on the Bragg spacing. This observation suggests that the arrangement of DNA strands in the complexes is determined by the structure of the polycation, and argues against suggestions that the structure of the collapsed state is maintained by the balance of long-range electrostatic repulsive and attractive forces. Instead we propose that DNA helices form a hexagonal array with counterions in the interstices between the helices resulting in a stable three-dimensional phase with high structural order. Arguments are presented favoring such a model in terms of stabilizing and destabilizing thermodynamic forces.     

X-ray diffraction studies of the contractile mechanism in single muscle fibres

The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X-ray diffraction. Changes in the intensities of the axial X-ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X-ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X-ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray diffraction studies of bacterial pili

A sample of bacterial pili was prepared from Escherichia coli. An X-ray diffraction pattern was obtained from an oriented wet gel specimen in 0.01 m-phosphate buffer (pH 7.0) packed in a capillary tube. Sixteen independent spots were observed with the spacing of the outermost being 4.2 Å. Analysis of the diffraction pattern shows that the arrangement of subunits in pili rods is strictly simple-helical with 3.14₅ units being present in one turn of the helix and the axial rise per unit being 8.09_o Å.     

X-ray diffraction studies on melanins in lyophylized melanosomes

A series of experiments was performed on lyophylized melanosomes in order to analyze the melanin in the natural state as polymerized into these organelles and to verify in such biological amorphous material the possibility of obtaining intensity scattering curves from which Bragg distances can be calculated. The results confirm the feasibility of the method and show that melanins in melanosomes maintain many structural features of the purified form and that the biochemical composition of the organelles can be responsible for the observed differences in the diffractograms. The presence in melanosomes of supramolecular paracrystalline aggregates was also clearly demonstrated.     

X-ray diffraction patterns from mammalian heart muscle

We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart.     

Time-resolved equatorial X-ray diffraction studies of skinned muscle fibres during stretch and release.

Equatorial X-ray diffraction patterns were recorded from small bundles of one to three chemically skinned frog sartorius muscle fibres (time resolution 250 microseconds) during rapid stretch and subsequent release. In the relaxed state, the dynamic A-band lattice spacing change as a result of a 2 % step stretch (determined from the positions of the 10 and 11 reflections) resulted in a 21 % increase in lattice volume, while static studies of spacing and sarcomere length indicated than an increase in volume of >/=50 % for the same length change. In rigor, stretch caused a lattice volume decrease which was reversed by a subsequent release. In activated fibres (pCa 4.5) exposed to 10 mM 2,3-butanedione 2-monoxime (BDM), stretch was accompanied by a lattice compression exceeding that of constant volume behaviour, but during tension recovery, compression was partially reversed to leave a net spacing change close to that observed in the relaxed fibre. In the relaxed state, spacing changes were correlated with the amplitude of the length step, while in rigor and BDM states, spacing changes correlated more closely with axial force. This behaviour is explicable in terms of two components of radial force, one due to structural constraints as seen in the relaxed state, and an additional component arising from cross-bridge formation. The ratio of axial to radial force for a single thick filament resulting from a length step was four in rigor and BDM, but close to unity for the relaxed state.