Abstract
Smooth myosin regulatory light chain (RLC) was exchanged with RLC labeled with benzophenone-4-iodoacetamide at Cys-108. Irradiation under conditions that favor the folded (10 S) conformation resulted in 10 S cross-linked myosin that could not unfold. Purified 10 S cross-linked myosin was cross-linked between the RLC of one head to light meromyosin between leucine 1554 and glutamate 1583, adjacent to a predicted noncoiled region, approximately 60 nm from the tip of the tail. At high ionic strength without actin, product release from one-half of the heads was slow (like 10 S) whereas the other half were activated. This suggests that tail binding to the RLC carboxyl-terminal domain stabilizes ionic interactions important to slow nucleotide release. With actin, product release from both (un)phosphorylated 10 S cross-linked myosin was from one slow population similar to unphosphorylated filaments. 10 S cross-linked myosin weakly bound actin (dissociation constant > 500 microM) and did not move actin in vitro. Single-headed myosin did not fold or trap nucleotide. These and other data suggest that "trapping" occurs only with both heads and the tail binds to a newly formed site, which includes the RLC carboxyl-terminal domain, once trapping has occurred.
Highlights
That additional proteins are required for the stabilization of myosin filaments in the presence of ATP [13, 14]
Preparation and Purification of 10 S Cross-linked Myosin— Initial studies of myosin exchanged with BP-regulatory light chain (RLC) were aimed at identifying interactions of the COOH-terminal domain of RLC with the rest of the myosin molecule
The presence of the RLC in the band that migrates above the free heavy chain (HC) on PAGE was confirmed by Western blot of the gel
Summary
That additional proteins are required for the stabilization of myosin filaments in the presence of ATP [13, 14]. The regulatory light chain is an essential component of the head-tail junction binding site for the tail because light chaindeficient myosin does not fold [17]. Ca2ϩ binding to the essential light chain activates the Mg2ϩATPase and destabilizes the 10 S conformation [6] These findings suggest that the 10 S myosin contains some of the structural aspects of inactive filaments. 10 S cross-linked myosin did not move actin filaments in an in vitro motility assay and bound weakly to actin By combining this information with a study of folding in single-headed myosin and the work of others, conclusions were drawn about the specific interactions of the tail with RLC and its relationship to the trapped state
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