Geoff R. Mant

Time‐resolved x‐ray diffraction station: X‐ray optics, detectors, and data acquisition

A new x‐ray beamline has recently been built, and is now operational, on dipole magnet 2 of the Synchrotron Radiation Source (SRS) at Daresbury. This beamline takes 32 mrad of horizontal aperture from a central tangent point. The time‐resolved x‐ray diffraction (TRXD) Station 2.1, takes 17 mrad of horizontal aperture and is the central point of this paper. Beamline 2 has been realized as part of a SERC–MRC agreement.

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

SummaryResults 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 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 the 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. The results of comparing the meridional intensities between the myosin layer lines at rest and the actomyosin layer lines at the plateau of tension (measured to a resolution of approximately 2.6 nm) are interpreted to indicate that the majority of the myosin heads in the actomyosin complex do not perform random axial rotations with a mean value greater than approximately 3.0 nm. From this we conclude that the extent of axial order in the interacting heads must be at least as high as is that of resting heads.

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

SummaryUsing the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8°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° at rest to ca 90° 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. To account for the diffraction data in terms of the conventional tilting head model one would have to suppose that a few of the heads, and/or a small part of their mass perform the much larger motions demanded by that model. Therefore we conclude either that the required information is not available in our patterns or that an alternative hypothesis for contraction has to be developed.

W16.1: A new fixed wavelength diffraction station at the SRS Daresbury

Station W16.1 is a fixed wavelength (1.4 A) x‐ray diffraction station recently constructed and commissioned at the SRS. It has been designed specifically for time‐resolved studies of noncrystalline and fibrous materials and optimized for low angle measurements. Wide angle diffraction will also be available with simultaneous small and wide angle scattering/diffraction a future facility. In order to perform dynamic (∼1 ms) low angle measurements on weakly scattering systems, the station design has had to incorporate several novel features so as to achieve the predicted 1×1013 photon/s at the specimen.

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.