A combined SAXS/WAXS investigation of the phase behaviour of di-polyenoic membrane lipids.

Real-time measurements of the SAXS/WAXS diffraction patterns of aqueous dispersions (1:1 wt/wt) of the di-polyenoic lipids di-18:2 PC, di-18:3 PC, di-18:2 PE and di-18:3 PE were made over the temperature range 10 degrees to about -80 degrees C. The results of these measurements were compared to similar measurements performed on the corresponding di-18:0 and di-18:1 derivatives. SAXS measurements of the temperature dependence of lamellar repeat distances show that the di-polyenoic lipids undergo broad second-order transitions between their gel and liquid-crystal lamellar phases spanning 30-40 degrees C. The di-18:1 and di-18:0 derivatives, in contrast, undergo abrupt first-order transitions. The gel phases of the di-18:0 derivatives are characterised by two-component WAXS patterns with a sharp component close to 0.42 nm and a broader component at narrower spacings. On cooling, these lipids appear to undergo an initial transition to an L beta, phase followed by a conversion to an Lc phase. The gel phases of the di-18:1 derivatives also show two-component patterns but with the sharp component centred closer to 0.44 nm. The di-polyenoic lipids, in contrast, are characterised by a single broad peak centred at a spacing of about 0.42 nm, close to that of conventional L beta phases. The changes in lamellar repeat distance accompanying the transitions in the di-monoenoic and di-polyenoic lipids, all of which occur in the frozen state, are very similar, indicating that the acyl chains of the polyenoic lipids are close to their maximum extension in the gel state. The WAXS patterns of the polyenoic lipids suggest that the saturated upper parts of the acyl chains are packed on a regular hexagonal lattice while their polyunsaturated termini remain relatively disordered.

The effect of temperature on the structure of vinblastine-induced polymers of purified tubulin: detection of a reversible conformational change.

Addition of the antimitotic drug vinblastine to solutions of purified tubulin induces the formation of helical polymers whose structure and type of aggregation is determined by the concentration of magnesium. While paracrystalline arrangements of single coils are observed at low concentrations of the ion, for concentrations higher than 6 mM free double-coiled spirals are obtained, which are indistinguishable from those obtained in the presence of microtubule-associated proteins (MAPs). This result is consistent with a similar effect of magnesium and MAPs in neutralizing negative charges on the tubulin molecule and so allowing for lateral contacts between protofilaments. The effects that temperature has on the structure of both types of polymers, free spirals or paracrystals, have been monitored using time-resolved X-ray solution scattering. This study shows that a temperature increase: (1) affects the length and lateral aggregation of the spirals in the paracrystalline sample; (2) induces a reversible increase of the helical pitch in both types of polymers that closely follows the temperature change; (3) produces an irreversible aggregation of some of the protein in both types of polymers; and (4) can induce a reversible transformation from one type of structure to the other when the concentration of Mg2+ is in the boundary between the two ranges. We suggest that the changes in pitch are due to a temperature-induced conformational change of the tubulin molecule. This effect may be related to the structural modifications that result in the temperature-induced assembly of microtubules in vitro under normal conditions of assembly.

X-ray evidence that in contracting live frog muscles there exist two distinct populations of myosin heads.

Using synchrotron radiation and whole muscles, 2 ms time-resolved x-ray diffraction patterns were recorded at 8 degrees C. The results show that in both isotonic and isometric contractions, as well as in length changes imposed at maximum tension [Po], the meridional third myosin layer line consists of two distinct reflections with different intensities and spacings that measure approximately 14,623 and 14,412 nm at Po. Although the intensity behavior of the two reflections is strikingly different during quick releases, it is very similar during stretches. Study of the time courses indicates that myosin heads diffracting at Po with the approximately 14.623 nm periodicity are actively involved in tension production. Those diffracting at Po with the periodicity of approximately 14.412 nm appear not be associated with tension production during isometric contraction and releases, but the results suggest that they are recruited during stretches and here contribute to tension production. Our most important conclusion is that under all conditions of contraction we have investigated there exist two populations of myosin heads, each with a well defined axial disposition and configuration.

Real-time simultaneous wide- and small-angle fibre diffraction.

A combination of two independent imaging area-detector systems controlled by a single data-acquisition system, provides a powerful system for X-ray diffraction studies of time-resolved phenomena over a wide q range, in samples with intrinsic or induced structural orientation. With this system we have observed a transient, tensile-stress-induced, orthorhombic-to-monoclinic transition in high-density polyethylene.

Computing for synchrotron radiation experiments.

Computing provision is of major concern to synchrotron radiation researchers. An overview is presented of the computing facilities for synchrotron radiation work at the Synchrotron Radiation Source, Daresbury Laboratory. Data acquisition, reduction and analysis are seen as an integrated activity essential for full utilization of beam time. We discuss the evolution of data-acquisition systems from stand-alone systems of limited computing power, to a unified project computing village using state-of-the-art equipment for combined-technique, high-data-rate experiments. A brief account of software packages for data reduction and analysis is also given.

Time-resolved X-ray diffraction studies of myosin head movements in live frog sartorius muscle during isometric and isotonic contractions.

Using the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8 degrees C were recorded with a time resolution of 2 or 4 ms. In isometric contractions tetanic peak tension (P0) is reached in ca 400 ms. Under such conditions, following stimulation from rest, the timing of changes in the major reflections (the 38.2 nm troponin reflection, and the 21.5 and 14.34/14.58 nm myosin reflections) can be explained in terms of four types of time courses: K1, K2, K3 and K4. The onset of K1 occurs immediately after stimulation, but that of K2, K3 and K4 is delayed by a latent period of ca 16 ms. Relative to the end of their own latent periods the half-times for K1, K2, K3 and K4 are 14-16, 16, 32 and 52 ms, respectively. In half-times, K1, K2, K3 lead tension rise by 52, 36 and 20 ms, respectively. K4 parallels the time course of tension rise. From an analysis of the data we conclude that K1 reflects thin filament activation which involves the troponin system; K2 arises from an order-disorder transition during which the register between the filaments is lost; K3 is due to the formation of an acto-myosin complex which (at P0) causes 70% or more of the heads to diffract with actin-based periodicities; and K4 is caused by a change in the axial orientation of the myosin heads (relative to thin filament axis) which is estimated to be from 65-70 degrees at rest to ca 90 degrees at P0. Isotonic contraction experiments showed that during shortening under a load of ca 0.27 P0, at least 85% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, whilst their axial orientation does not change from that at rest. During shortening under a negligible load, at most 5-10% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, and their axial orientation also remains the same as that at rest. This suggests that in isometric contractions the change in axial orientation is not the cause of active tension production, but rather the result of it. Analysis of the data reveals that independent of load, the extent of asynchronous axial motions executed by most of the cycling heads is no more than 0.5-0.65 nm greater than at rest.(ABSTRACT TRUNCATED AT 400 WORDS)

Two-dimensional time-resolved X-ray diffraction studies of live isometrically contracting frog sartorius muscle.

Results were obtained from contracting frog muscles by collecting high quality time-resolved, two-dimensional, X-ray diffraction patterns at the British Synchrotron Radiation Source (SERC, Daresbury, Laboratory). The structural transitions associated with isometric tension generation were recorded under conditions in which the three-dimensional order characteristic of the rest state is either present or absent. In both cases, new layer lines appear during tension generation, subsequent to changes from activation events in the thin filaments. Compared with the 'decorated' actin layer lines of the rigor state, the spacings of the new layer lines are similar whereas their intensities differ substantially. We conclude that in contracting muscle an actomyosin complex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from rest to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as arising from axial disposition of the interacting myosin heads in the actomyosin complex. Model calculations are presented which support this view. We argue that in a situation where an actomyosin complex is formed during contraction, one cannot describe the diffraction features as being either thick or thin filament based. Accordingly, the layer lines seen during tension generation are referred to as actomyosin layer lines. It is shown that these layer lines can be indexed as submultiples of a minimum axial repeat of approximately 218.7 nm. After lattice disorder effects are taken into account, the intensity increases on the 15th and 21st AM layer lines at spacings of approximately 14.58 and 10.4 nm respectively, show the same time course as tension rise. However, the time course of the intensity increase of the other actomyosin layer lines and of the spacing change (which is the same for both phenomena) shows a substantial lead over tension rise. These findings suggest that the actomyosin complex formed prior to tension rise is a non-tension-generating state and that this is followed by a transition of the complex to a tension-generating state. The intensity increase in the 15th actomyosin layer line, which parallels tension rise, can be accounted for assuming that in the tension-generating state the attached heads adopt (axially) a more perpendicular orientation with respect to the muscle axis than is seen at rest or in the non-tension-generating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative study of the supramolecular structure of frog sartorius and dorsal semitendinosus muscle.

This report describes a comparative X-ray diffraction study of the supramolecular structure of frog sartorius and semitendinosus muscles. For sarcomere lengths of 2.7 microns and below the X-ray diffraction diagrams of each muscle type are very similar; the only differences being that the diffraction diagram for semitendinosus muscles exhibit the presence of a broad diffraction band or a cluster of diffraction orders at a spacing of ca. 230.0 nm and, also, they lack a periodicity of ca. 102.0 nm. For sarcomere lengths greater than 2.7 microns disruption of the sarcomere from sartorius muscle occurs as seen by the loss of sampling in the diffraction diagram. The semitendinosus muscle can be stretched to much longer lengths (in excess of 3.0 microns) before a loss of sampling is detected. The data also shows that in the case of the semitendinosus muscle for long sarcomere lengths transverse bands of mass are able to move without retaining a defined distance to either the Z or the M lines. This is not observed in the case of the sartorius muscle. Thus, at resolutions between ca. 3.6 microns and 7.50 nm significant ultrastructural differences between these two muscles are apparent. The data suggest that the ability of these mass bands to move may be responsible for the differences in the development of passive tension exhibited by these two muscles.

Two-dimensional time resolved X-ray diffraction of muscle: recent results.

This report provides a preliminary sketch of the results obtained in a two-dimensional time resolved X-ray diffraction study of "live" frog sartorius muscles undergoing isometric tetani. These results demonstrate the recently developed capability to record time resolved (10 msec time resolution), two-dimensional X-ray diffraction diagrams throughout the cycle of contraction. The correlation between the time courses of the diffraction features in the whole of the diffraction diagram establishes a sequence of structural events, which suggest that during the transition from rest to the plateau of tension and the subsequent recovery, the following sequence of events takes place: a) Following the activation phase, which is best monitored by the increase of intensity on the second actin layer line at 18.0 nm spacing (5), there is the onset of three dimensional disorder due to the filaments losing their axial alignment and the myosin heads rotating azimuthally and moving radially outwards. A set of low-angle layer lines, following the actin based spacings seen in rigor (i.e., at spacings of ca. 36.5-37.5, 24.0 and 18.0 nm) become visible and those at ca. 24.0 and 18.0 nm appear to increase in intensity during this phase with a time course that cannot be determined accurately because of the proximity of the neighbouring first, second and third myosin layer lines and the weakness of these diffraction features. Whether the first of these layer lines increases or not is difficult to ascertain. Moreover, proper account of the loss in crystallinity during the development of tension must be made before the comparisons in intensity between the rest and peak of tension states have any significance. Nevertheless, these features together with the behaviour of the equatorial reflections and the meridional region of the third myosin layer line indicate that a sizeable fraction of the crossbridges may become axially disposed with an actin based periodicity. The formation of this complex does not immediately result in the generation of tension. The labelling of the thin filaments is also reflected in the main actin layer lines at 5.9 and 5.1 nm. b) The tension generating phase is monitored by the intensity changes in the meridional region of the third myosin layer line, which are best explained by a change in the orientation/conformation of the tension bearing crossbridges, (which probably adopt a more perpendicular orientation to the filament axis). c) At the end of stimulation, the crossbridges return to an axial spacing and axial orientation (although not yet azimuthal) similar to the one at rest.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle.

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.