Structural characterization of weakly attached cross-bridges in the A*M*ATP state in permeabilized rabbit psoas muscle.
It is well established that in a skeletal muscle under relaxing conditions, cross-bridges exist in a mixture of four weak binding states in equilibrium (A*M*ATP, A*M*ADP*P(i), M*ATP, and M*ADP*P(i)). It has been shown that these four weak binding states are in the pathway to force generation. In the past their structural, biochemical, and mechanical properties have been characterized as a group. However, it was shown that the myosin heads in the M*ATP state exhibited a disordered distribution along the thick filament, while in the M*ADP*P(i) state they were well ordered. It follows that the structures of the weakly attached states of A*M*ATP and A*M*ADP*P(i) could well be different. Individual structures of the two attached states could not be assigned because protocol for isolating the two states has not been available until recently. In the present study, muscle fibers are reacted with N-phenylmaleimide such that ATP hydrolysis is inhibited, i.e., the cross-bridge population under relaxing conditions is distributed only between the two states of M*ATP and A*M*ATP. Two-dimensional x-ray diffraction was applied to determine the structural characteristics of the attached A*M*ATP state. Because the detached state of M*ATP is disordered and does not contribute to layer line intensities, changes as a result of increasing attachment in the A*M*ATP state are attributable to that state alone. The equilibrium toward the attached state was achieved by lowering the ionic strength. The results show that upon attachment, both the myosin and the first actin associated layer lines increased intensities, while the sixth actin layer line was not significantly affected. However, the intensities remain weak despite substantial attachment. The results, together with modeling (see J. Gu, S. Xu and L. C. Yu, 2002, Biophys. J. 82:2123-2133), suggest that there is a wide range of orientation of the attached A*M*ATP cross-bridges while the myosin heads maintain some degree of helical distribution on the thick filament, suggesting a high degree of flexibility in the actomyosin complex. Furthermore, the lack of sensitivity of the sixth actin layer line suggests that the binding site on actin differs from the putative site for rigor binding. The significance of the flexibility in the A*M*ATP complex in the process of force generation is discussed.
Xu
S
S
National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. xus@exchange.nih.gov
Gu
J
J
Melvin
G
G
Yu
L C
LC
D000249:Adenosine Monophosphate / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D006868:Hydrolysis; D007477:Ions; D008301:Maleimides / Q000737:chemistry; D008956:Models, Chemical; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D016658:Psoas Muscles / Q000378:metabolism*; D011817:Rabbits; D013696:Temperature; D014965:X-Rays
D007477:Ions; D008301:Maleimides; D000249:Adenosine Monophosphate; C028525:N-phenylmaleimide; D000251:Adenosine Triphosphatases; D009218:Myosins