We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorderto-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.EPR ͉ regulation ͉ structure S mooth muscle contraction is activated by phosphorylation of Ser-19 on myosin regulatory light chain (RLC) (1). The goal of the present study is to elucidate the structural changes that are induced in RLC upon phosphorylation. Several essential structural features of this regulatory process have been identified. First, regulation by phosphorylation requires two myosin heads, because both S1 and single-headed myosin are constitutively active (2, 3). A portion of the ␣-helical coiled-coil tail (S2) region appears also to be required for regulation (4). Thus the twoheaded heavy meromyosin (HMM) fragment has all of the features that are needed for full regulation.What structural elements of RLC are important for regulation? Chimeras containing the C terminus from skeletal RLC and the N terminus from smooth RLC remain in the ''off'' state even after phosphorylation, suggesting that the C-terminal domain of RLC is important for regulation (5). Mutational studies suggest that the ''linker helix,'' which connects the N-and C-terminal lobes of RLC, is crucial for regulation (6). The N-terminal 24 aa of RLC, which include the sites of phosphorylation (Thr-18, Ser-19), are proposed to play an important role in RLC function (7-9), but there is no structural information about this N-termi...