In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref. 1). The role of these light chains in vertebrate skeletal muscle myosin has remained obscure. Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement. We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity. Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity. We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement.
Smooth muscle myosin filaments formed in 0.15 M KCI are depolymerized by MgATP to a lOS component, rather than to the 6S component typical of myosin monomer in high salt concentrations. This lOS species is also monomeric as determined by sedimentation equilibrium and calculated from the diffusion and sedimentation coefficients. The conformation of lOS myosin is, however, very different from that of 6S myosin, which has a flexible but extended rod. The Stokes radius and the viscosity of lOS myosin are less than those of 6S myosin, consistent with a structure in which the rod is bent. Electron microscopy of rotaryshadowed preparations confirmed that the light meromyosin region of the rod is bent back on subfragment 2, that region of the rod adjacent to the two globular heads. MgATP and dephosphorylation of the 20,000 molecular weight light chain increase the amount of 10S myosin present in 0.15 M KCI; addition of salt converts lOS myosin back to the typical 6S conformation. We conclude that smooth muscle myosin preferentially forms a bent or folded conformation instead ofthe extended shape usually associated with skeletal muscle myosin, provided that the salt concentration is kept sufficiently low. Here we show that 10S myosin is not a dimer, but a monomer in which the rod is folded back upon itself such that the light meromyosin (LMM) region interacts with its own subfragment-2 (S-2) region. Addition of salt reforms the 6S conformation in which the rod is extended. The salt concentration thus has a large effect on the conformation of monomeric smooth muscle myosin, with this unusual bent form occurring at physiological ionic strength. MATERIALS AND METHODSPreparation of Calf Aorta Myosin. Myosin was prepared as described by Megerman and Lowey (7). To minimize phosphorylation of myosin by light chain kinase, 1 mM EGTA was added to all buffers, and MgATP was added to the crude myosin just prior to gel filtration. In this way, myosin was isolated with <10% phosphorylation. Myosin concentration was determined from Al% = 4.8.Gel Electrophoresis. NaDodSO4/polyacrylamide gel electrophoresis was performed according to the method ofLaemmli (8), with a 5-20% gradient ofacrylamide and a 4% stacking gel. Crosslinked myosin was separated on 2.5% acrylamide/0.5% agarose slab gels in the Laemmli buffer system. Phosphorylated and dephosphorylated LC20s were separated on glycerol/ acrylamide gels. The running gel contained 40% (vol/vol) glycerol and 10% acrylamide; the stacking gel, 40% glycerol and 3.5% acrylamide. The running and gel buffers were 20 mM Tris/ glycine, pH 8.6 (9). Myosin samples were applied to the gel in 30 mM Tris/glycine, pH 8.6/8 M urea/0.2 mM EDTA/0.1% 2-mercaptoethanol.Crosslinldng. A 20 mg/ml solution ofdimethyl suberimidate was prepared immediately before use. Myosin at 1 mg/ml (10 mM KPi, pH 7.5/5 mM MgCl2/1 mM EGTA/1 mM MgATP/ 0.15 or 0.6 M KCl) was crosslinked by addition of dimethyl suberimidate at 6 mg/ml for 1 hr at room temperature. As determined by sedimentation velocity, crosslinked 10S ...
ATP hydrolysis drives actin filament dynamics. The effect known as treadmilling arises from the preferential addition of ATP-actin monomers to the plus or barbed end of actin and the preferential dissociation of ADP-actin from the minus or pointed end. Hydrolysis of ATP occurs after the monomer is incorporated into the filament. In addition, actin-binding proteins often have significantly different affinities for the ADPbound versus ATP-bound forms of actin. Both of these lines of evidence strongly suggest that there must be significant structural changes in actin induced by ATP hydrolysis. Indirect solution techniques such as proteolytic digestion rates and fluorescence studies (reviewed in Ref. 1) are in agreement with conformational differences between the ADP and ATP states, but direct structural evidence has been lacking until recently.Based on crystal structures of a tetramethylrhodaminelabeled monomeric actin (TMR-actin) 2 with ADP (2) or AMP-PNP (3) at the active site, Dominguez and co-workers proposed the provocative idea that ATP hydrolysis initiates a series of changes originating at the active site, that ultimately cause a loop-to-helix transition in the DNase binding loop in subdomain 2 of actin ("D-loop," residues in subdomain 2 of actin which comprise part of the DNase I binding site). They suggested that this was the long sought after change in structure between ADP and ATP actin. The cleft between subdomains 2 and 4 of actin remained closed in both nucleotide states. Despite this observation, an opposing point of view (4) held that the more important conformational change is an opening of the cleft upon ATP hydrolysis, a change that would be compatible with that observed for other nucleotide hydrolyzing proteins. Docking of actin crystal structures into negatively stained images of F-actin was also consistent with the idea that ADPactin had a more open cleft than the triphosphate state (5). If the latter view is correct, one would have to suppose that the modification of Cys 374 by TMR stabilized the closed conformation and inactivated the nucleotide sensing mechanism by virtue of the binding of rhodamine between subdomains 1 and 3, a potential hinge region (6) of the molecule. It has been suggested (4) that the short helix observed in the crystal structure of the ADP state of TMR-actin could be formed as a result of contacts with neighboring molecules in the crystal. We provide further evidence to support this idea, and suggest a pathway through which nucleotide-dependent changes may propagate through fortuitous crystal packing interactions from one monomer to the D-loop region of another in the TMR-actin crystals.Here we crystallized and expressed a cytoplasmic actin that was rendered incapable of polymerization by virtue of two surface mutations in subdomain 4 (A204E/P243K). This strategy negates concerns raised about the TMR modification of actin.De novo crystallization of AP-actin with either ATP or ADP at the active site reveals obligatory nucleotide-dependent confor-* This work was suppor...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.