2-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 genera tion were recorded under conditions in which the three-dimensional ord er characteristic of the rest state is either present or absent. In bo th cases, new layer lines appear during tension generation, subsequent to changes from activation events in the thin filaments. Compared wit h the 'decorated' actin layer lines of the rigor state, the spacings o f the new layer lines are similar whereas their intensities differ sub stantially. We conclude that in contracting muscle an actomyosin compl ex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from res t to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as a rising from the axial disposition of the interacting myosin heads in t he 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 featur es as being either thick or thin filament based. Accordingly, the laye r lines seen during tension generation are referred to as actomyosin l ayer lines. It is shown that these layer lines can be indexed as submu ltiples of a minimum axial repeat of approximately 218.7 nm. After lat tice 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 actomy osin 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 transitio n 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 att ached heads adopt (axially) a more perpendicular orientation with resp ect to the muscle axis than is seen at rest or in the non-tension-gene rating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations. The results of compar ing 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 run) are interpreted to indicate tha t the majority of the myosin heads in the actomyosin complex do not pe rform random axial rotations with a mean value greater than approximat ely 3.0 nm. From this we conclude that the extent of axial order in th e interacting heads must be at least as high as is that of resting hea ds.