Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.Smooth muscle myosin (SMM) 1 and nonmuscle myosin are hexameric motor ATPases of ϳ500 kDa mass composed of pairs of HC, ELC, and RLC. A globular motor domain (N-terminal HC) and a light chain binding domain (HC ϩ ELC ϩ RLC) form a head (S1), and the two heads are dimerized by the HC C-terminal halves, which form a coiled-coil. The motor domain contains the catalytic site and the actin binding site (1). Only the head domain and its subfragments have been crystallized to date so there are no atomic resolution structures of a doubleheaded construct.The actin-activated ATPase activity and motor properties of SMM and nonmuscle myosin are regulated by phosphorylation of Ser-19 of the RLC, which is greater than 10 nm from the catalytic site (1-4). Phosphorylation enhances the ATPase activity by more than 1000-fold at saturating actin concentrations (5, 6). Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM and nonmuscle myosin. HMM, which lacks the C-terminal two-thirds of the tail, is double-headed and regulated (5-7), but expressed HMM constructs with truncated tails too short to form stable double-headed structures are unregulated (8 -10) as is S1 (7,11,12) and single-headed myosin (13,14). Furthermore, a nonmuscle HMM construct without one of the motor domains but with both regulatory domains is not regulated (15). Therefore, two full heads connected together with enough coiled-coil to allow for dimerization are required for regulation.Removal of the RLC abolishes regulation (16), but the ELC can be removed with re...