Site-directed spin labeling EPR (SDSL-EPRtroponin I ͉ spin labels ͉ Fourier transform electron paramagnetic resonance ͉ DEER ͉ dipolar R egulation of striated muscle contraction is associated with Ca 2ϩ -dependent structural transitions in the muscle thin filament, which is composed of the troponin complex, tropomyosin, and actin. Troponin is composed of three components: TnC, which binds Ca 2ϩ ; TnI, which inhibits actomyosin activity; and TnT, which anchors TnC and TnI to tropomyosin. Muscle contraction is initiated by the binding of Ca 2ϩ to the N-lobe regulatory sites of TnC. The N lobe then undergoes a structural transition from a closed to an open form, which then facilitates the release of the inhibitory region of TnI from actin, binding to TnC and stimulation of the actomyosin ATPase. The mechanism of this signaling pathway is still tentative (for review, see ref. 1).Crystal and NMR structures are available for TnC and several complexes of TnC with TnI fragments. The crystal structure of TnC reveals a dumbbell-shaped protein consisting of two globular domains, joined by a 22-residue central ␣-helix (2-4). Each domain contains a hydrophobic cleft and two helix-loop-helix EF-hand metal binding motifs; two high-affinity Ca 2ϩ ͞Mg 2ϩ sites in the C-terminal domain (sites III and IV) and two low-affinity Ca 2ϩ specific sites in the N-terminal domain (sites I and II). In skeletal TnC, sites III and IV are permanently occupied by Mg 2ϩ and facilitate the structural binding of TnC to the contractile apparatus (5). Binding of Ca 2ϩ to sites I and II is the physiological trigger for muscle contraction. In cardiac TnC, binding site I is inactive and Ca 2ϩ binding to site II does not induce as large a structural change as observed in skeletal TnC (6). TnI is capable of inhibiting actomyosin ATPase in the absence of other subunits, but Ca 2ϩ -dependent regulation requires TnC, TnT, and tropomyosin. The inhibitory region (skTnI 96-117) alone can fully inhibit actomyosin ATPase activity (7) possibly by binding either to actin or to TnC in the ''on'' or ''off'' states (8-10). The corresponding residues for the cardiac inhibitory region are 129-150 because of a unique Ϸ32 residue N-terminal extension of cTnI.Structural information for the intact troponin complex is limited to low-resolution neutron diffraction and electron microscopy studies (11-13), though several high-resolution structures of TnC with bound TnI peptides are available. Two computational models have been recently proposed for the binary complex of TnC and TnI (14,15). Both models have TnI and TnC in an antiparallel arrangement with multiple interaction sites between the two subunits. NMR, crystallography, and neutron scattering was used by Tung et al. (15) to develop a computational model of the binary complex in which TnI winds around TnC in either a left-handed manner (model ''L'') or a right-handed manner (model ''R''). In both structures, the inhibitory region of TnI is modeled as a flexible ␤-hairpin in close proximity to the central helix of TnC. ...