Structural Characterization of the Binding of Myosin·ADP·P_i to Actin in Permeabilized Rabbit Psoas Muscle

¹ NIH
² East Virginia Medical School, Norfolk, VA

^* To whom correspondence should be addressed. E-mail: lcyu{at}helix.nih.gov.

Submitted on April 12, 2006
Revised on May 24, 2006
Accepted on 30 June 2006

	Abstract

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A·M·ADP and A·M) and the weakly bound A·M·ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereo-specific" attachment) and the attached heads exhibit little spatial fluctuation. In the pre-hydrolysis weakly bound A·M·ATP state, the orientation of the attached myosin heads assumes a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head (1,2). The structure of the other weakly bound state A·M·ADP·P_i is, however, poorly understood. This state is thought to be the critical pre-power stroke state, poised to make the transition to the strong binding, force generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A·M·ADP·P_i state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weak binding M·ATP, M·ADP·P_i states and the weakly attached A·M·ATP state, (2,3,1,4,5) in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A·M·ADP·P_i. The present series of experiments were carried out under relaxing condition at 25°C, where ~95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, while the intensity profiles did not change. The configuration (mode) of attachment in the A·M·ADP·P_i state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g. reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M·ADP·P_i with strongly coupled domains may contribute to the unique attachment configuration -- the "primed" myosin heads may function as "transient struts" when attaches to the thin filaments.

Key Words: Effects of PEG on actin-myosin binding, X-ray diffraction of skeletal muscle, actin filaments in muscle, myosin filaments in muscle, order-disorder in thick filaments