Structural Characterization of Weakly Attached Cross-Bridges in the A·M·ATP State in Permeabilized Rabbit Psoas Muscle

Abstract

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.

Insight into the actin-myosin motor from x-ray diffraction on muscle

“…On the other hand, there is evidence for a substantial contribution of weakly bound myosin heads with either ATP (88) or ADP⋅P i (89) in their active sites to the off-meridional intensity of the myosin layer lines. To dissect the contribution of weakly bound heads from that of detached ones, x-ray diffraction patterns from muscle fibers with permeabilized cell membrane in the absence of Ca 2+ were compared at different sarcomere length, temperature, ionic strength, in the absence and in the presence of polyethylene glycol that promotes weak binding.…”

“…To dissect the contribution of weakly bound heads from that of detached ones, x-ray diffraction patterns from muscle fibers with permeabilized cell membrane in the absence of Ca 2+ were compared at different sarcomere length, temperature, ionic strength, in the absence and in the presence of polyethylene glycol that promotes weak binding. Modeling of x-ray diffraction patterns of muscles with weakly bound myosin heads was performed (90) to obtain a quantitative description of the experimental data (88). The modeling suggests that weak binding of myosin heads to actin with a ~70° full range of dispersion of axial attachment angles and a ~150° full range of dispersion of azimuthal attachment angles fits the intensity profiles for myosin layer lines from M1 to M6.…”

“…The effect of weak binding of myosin heads to actin on the intensities of the actin layer lines is not well understood. An increase in the A1 layer line intensity, I A1 , was shown to occur upon a decrease in the ionic strength of the relaxing solution bathing permeabilized muscle fibers treated with Nphenylmaleimide (88). The treatment stops ATP hydrolysis and leaves myosin heads in a state where only weak binding to actin is possible while low salt was shown to promote weak binding (97,98).…”

“…The treatment stops ATP hydrolysis and leaves myosin heads in a state where only weak binding to actin is possible while low salt was shown to promote weak binding (97,98). On the other hand no increase in I A6 upon weak binding was found in experiments (88), suggesting that weak binding to actin is non-stereo-specific (99,88,90). Such binding is characterized by a range of azimuthal and axial angles of attachment of myosin heads to actin.…”

Approaches to physical fitness and sports medicine through X-ray diffraction analysis of striated muscle

“…For example, the intensity of MLL1 can be changed by manipulating the experimental solution to mimic or reproduce physiologically important states. When a mammalian skeletal muscle fiber is treated by crosslinking agents such as N-phenylmaleimide or cooled, MLL1 is weakened; and the peak position, which is reciprocally related to the gyration radius of the helix formed by the myosin heads, shifts toward the meridian [25][26][27][28] . Since crosslinking and cooling shift the chemical state of myosin heads from the M-ADP-Pi state to the M-ATP state, the observed decrease in the intensity and peak shift of the MLL1 is considered to indicate that myosin heads in the M-ATP are released from the rod and disordered.…”