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We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys(133) of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca(2+) state, the TnC44-TnI133-T distance was 42 A, with a narrow distribution (half-width of 9 A), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 A (width of 12 A) and 22 A (width of 3.4 A), respectively. These values were all consistent with recently published crystal structure (Vinogradova, M. V., Stone, D. B., Malanina, G. G., Karatzaferi, C., Cooke, R., Mendelson, R. A., and Fletterick, R. J. (2005) Proc. Natl Acad. Sci. U.S.A. 102, 5038-5043). Similar distances were obtained with the same spin pairs on a reconstituted thin filament in the +Ca(2+) state. In the -Ca(2+) state, the distances displayed broad distributions, suggesting that the regulatory region of TnI was physically released from the N-lobe of TnC and consequently fluctuated over a variety of distances on a large scale (20-80 A). The interspin distance appeared longer on the filament than on troponin alone, consistent with the ability of the region to bind actin. These results support a concept that the regulatory region of TnI, as a molecular switch, binds to the exposed hydrophobic patch of TnC and traps the inhibitory region of TnI away from actin in Ca(2+) activation of muscle.

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Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, approximately 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5'-(beta,gamma-imido)triphosphate and adenosine 5'-(gamma-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered. We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.

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We have measured the intersite distance, side-chain mobility and orientation of specific site(s) of troponin (Tn) complex on the thin filaments or in muscle fibres as well as in solution by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). We have examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a human cardiac (hc)TnC monomer state and hcTnC-hcTnI binary complex. An interspin distance between G42C (B helix) and C84 (D helix) was 18.4 angstroms in the absence of Ca2+. The distance between Q58C (C helix) and C84 (D helix) was 18.3 angstroms. Distance changes were observed by the addition of Ca2+ and by the formation of a complex with TnI. Both Ca2+ and TnI are essential for the full opening -3 angstroms of the N-domain in cardiac TnC. We have determined the in situ distances between C35 and C84 by measuring pulsed electron-electron double resonance (PELDOR) spectroscopy. The distances were 26.0 and 27.2 A in the monomer state and in reconstituted fibres, respectively. The addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, indicating that Ca2+ binding to the N-lobe of hcTnC induced a larger structural change in muscle fibres than in the monomer state. We also succeeded in synthesizing a new bifunctional spin labels that is firmly fixed on a central E-helix (94C-101C) of skeletal(sk)TnC to examine its orientation in reconstituted muscle fibres. EPR spectrum showed that this helix is disordered with respect to the filament axis. We have studied the calcium structural transition in skTnI and tropomyosin on the filament by SDSL-EPR. The spin label at a TnI switch segment (C133) showed three motional states depending on Ca2+ and actin. The data suggested that the TnI switch segment binds to TnC N-lobe in +Ca2+ state, and that in -Ca2+ state it is free in TnC-I-T complex alone while fixed to actin in the reconstituted thin filaments. In contrast, the side chain spin labels along the entire tropomyosin molecule showed no Ca(2+)-induced mobility changes.

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We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.

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We have studied biological nano-machines, motor and switch proteins operating as supramolecular complexes by electron spin resonance (ESR) and found key features of their molecular movements. In all the systems, the specific movements of elements or domains were detected and quite dynamic at nanometer scale. We have observed two broad but distinct orientations, separated by a 25 degrees axial rotation, of a spin label attached specifically to the light chain (LC) domain of myosin motor in the muscle fibers. The distribution became only narrower upon muscle activation. ESR spectrum from the spin label of the neck-linker of dimeric kinesin motor consisted of immobilized and mobilized components and did not exhibit nucleotide-dependent mobility change. The distance between two labels of kinesin dimer was also measured by spin dipole-dipole interaction, showing a broad distribution and a nucleotide-dependent change on the nanometer scale (>1.5 nm). These results suggest that two LC domains of myosin and two neck linkers of kinesin play a similar role for sliding movement using two conformations. The spin label of the skeletal (Tn)-I regulatory domain (TnIreg) showed a large mobility change by Ca2+ ion suggesting a Ca-induced switch movement of TnIreg. Spin dipole-dipole interaction showed that in reconstituted muscle fibers both skeletal and cardiac TnC undergo Ca2+-induced structural change that is thought to be essential for TnIreg movement. We also succeeded in fixing the newly-synthesized bifunctional spin label rigidly on the TnC molecule in solution, indicating that we can determine the precise coordinate of the spin principal axis of troponin on the oriented filament.

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The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca(2+)-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32ns to 13ns upon Ca(2+)-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16ns) and immobile (35ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. We determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstroms and 27.2 angstroms in the monomer state and in fibres, respectively, and the addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state.

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An enantiomeric pair of C2-chiral bifunctionalised spin labels having a pyrrolidine nitroxide moiety, whose configurations were determined by X-ray crystal diffraction analysis, was prepared and applied to troponin C whose binding mode of double disulfide linkage was proved by EPR spectroscopy.

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Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.

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Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.

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Using electron spin resonance, we have studied dynamic structures of myosin neck domain and troponin C by site-directed spin labeling. We observed two broad but distinct orientations of a spin label attached specifically to a single cysteine (cys156) on the regulatoy light chain (RLC) of myosin in relaxed skeletal muscle fibers. The two probe orientations, separated by a 25 degrees axial rotation, did not change upon muscle activation, but orientational distributions became narrower substantially, indicating that a fraction of myosin heads undergoes a disorder-to-order transition of the myosin light chain domain upon force generation and muscle contraction. These results provide insight into the mechanism how myosin heads move their domains to translocate an actin filament. Site-directed spin-labeling was achieved by cysteine residues of human cardiac troponin C (TnC). Spin dipole-dipole interaction showed that free TnC undergoes a global structural change (extended-to-compact) by Ca2+ or Mg2+. The spectra from the spin labels at N-terminal half domain were broad and almost identical in parallel and perpendicular orientations of fiber, suggesting that the N-terminal of TnC molecule is flexible or disoriented with respect to the filament axis. We also succeeded, for the first time, in fixing the newly-synthesized bifunctional spin label rigidly on TnC molecule in solution (either in +/- Ca2+), giving a promise that we can determine the precise coordinate of the spin principal axis on protein surface.

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