Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin.
An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.
Marriott
G
G
Biomolecular and Cellular Dynamics Research Group, Max Planck Institute for Biochemistry, Martinsried, Germany.
Heidecker
M
M
D000199:Actins / Q000378:metabolism* / Q000494:pharmacology / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000840:Anisoles / Q000737:chemistry*; D004789:Enzyme Activation; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000199:Actins; D000840:Anisoles; D015879:Myosin Subfragments; C098542:1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene; D000251:Adenosine Triphosphatases