Communications Between the Nuclotide-and Actin-Binding Site of the Myosin Head in Muscle Fibers

Abstract

2′(or 3′)-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP), a fluorescent analog of ATP, and the derivatives were used to fluorescently label the myosin head in skinned skeletal muscle fibers. It has been known that a secondary site for TNP-ADP other than the active site exists in myosin S1. We found by fluorescence resonance energy transfer between mant ATP and TNP-AMP that the secondary site for TNP-nucleotides is located within 2 nm of the active site in skeletal muscle fibers. The changes in fluorescence intensity of muscle fibers in 20 μM TNP-AMP when nucleotides are bound may reflect changes of the structure of the active site of myosin heads. It was also shown that actin affected the extent of the fluorescence changes induced by ATP binding to the active site. Both ATP and caged ATP affected the fluorescence intensity, thus caged ATP interacts with the active site. When ATP was released from caged ATP by pulse photolysis in muscle fibers in TNP-AMP showed a transient increase in fluorescence intensity, and still greater fluorescence signal can be detected when the fiber actively contracted when Ca²⁺ was present.