The light chain binding domain of expressed smooth muscle heavy meromyosinacts as a mechanical lever

Structural data led to the proposal that the molecular motor myosin moves a ctin 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 he avy 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 o f unitary displacements and forces in the laser trap assay, Two shorter nec ked constructs had smaller unitary step sizes and moved actin more slowly t han WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold incre ase in the unitary step size compared with its control. Kinetic changes wer e also observed with several of the constructs, The mutant lacking a neck p roduced force at a somewhat reduced level, while the force exerted by the g iraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid le ver, with the fulcrum for movement and force located at a point within the motor domain.