The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever.
AUTHORS
- PMID: 10945998[PubMed].
ABSTRACT
Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or “neck.” To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.
Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or “neck.” To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.