Piotr G. Fajer, Drazen Raucher, Bishow Adhikari and Liz Fajer
National High Magnetic Field Lab & Dept. of Biology, Florida State University, Tallahassee
To determine the pre-power stroke conformation, we arrested the contractile cycle in AlF3 (+ATP/Ca). In this state the myosin heads are strongly bound to the thin filaments, as measured by mechanical stiffness and, independently, by the myosin head infusion assay. However, these bound heads do not generate active force. These trapped crossbridges are as disordered as relaxed (detached) heads, but their microsecond dynamics are significantly restricted. Modeling of the immobile fraction in terms of the number of attached heads, as estimated from stiffness, suggests that the bound heads rotate with a correlation time tr=150~400 ms. This is 50 - 100 times slower than the heads in relaxed fibers. Fluorescence energy transfer (FRET) between cys-707 (AEDANS) and the myosin nucleotide binding site (TNP.ADP) or the cys-374 of actin (AF) all indicate a different distribution of distances in the A.S1.ADP. AlF4- ternary complex and the A.S1.ADP (A.S1).
Post-power stroke states are approximated by the rigor ADP state. The global orientation and mobility of the myosin heads was found to be nearly identical to that of the rigor heads. We did, however, observe substantial internal domain reorganization on the addition of ADP. The novel a-iodoketo spin label revealed two different populations, both of which are distinct from those at the beginning (relaxation) and at the end (rigor) of the cycle.
The above orientational rearrangement was accompanied by the onset of large-amplitude microsecond motions, as measured by saturation-transfer EPR, with rates intermediate (tr = 150 ms) between the rotations of detached (tr = 7 ms) and rigor (tr = 2,500 ms) heads. Since other spin labels (MSL and IASL) report immobilization and a high degree of order, IKSL disordering must be limited to the probed domain, whereas MSL and IASL put an upper limit on the global order and mobility of the head. The implication of this finding is that the myosin head in a single chemical state (AMlADP) is capable of attaining more than one internal configuration. The presence of slow structural fluctuations is related to the slow release of the hydrolysis products of actomyosin ATPase.
Combining these and our earlier findings of the head orientation and dynamics we propose a sequence of events comprising contractile cycle: (a) relaxed heads are detached and free to execute large amplitude microsecond motions; (b) initial attachment, which occurs in the absence of Ca, restricts slightly (2-fold) the rate of motion, with equally small effects on the amplitude; (c) Ca activation of the thin filaments leaves the heads dynamically disordered, similar to weak binding; (d) the pre-power stroke state(s) are more immobilized, but still not stereospecifically bound, they are statically disordered; (e) following force generation all the heads are stereospecifically bound to actin, but there is significant internal domain mobility associated with the presence of nucleotide in the active site; (f) the release of ADP inhibits all internal motions.
We believe that as the head progresses through the contractile cycle, its mobility is restricted by the formation of stronger interactions with actin. However, even when the binding is strong, the heads exhibit a wide range of orientations with respect to the actin filament. The disorder-to-order transition near the end of the cycle results in the generation of strain (force) between the filaments, which cannot be relieved since the heads are strongly bound to actin. Local domain motions in the vicinity of the active site, Cys-707, are present after force generation, facilitating the protein flexibility necessary to release the ATP hydrolysis products.