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We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics.

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.

We have used electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to measure structural transitions of the catalytic domain (CD) in Dictyostelium myosin II. The use of BSL is a critical feature in this work. The bifunctional attachment of BSL eliminates most of the nanosecond motions characteristic of monofunctional labels, making it possible to measure protein structural transitions with a precision not previously achievable. Two double-Cys constructs were engineered with Cys residues at helical locations i and i+4 (494.498 and 639.643). Residues 494.498 are located on the relay helix and residues 639.643 are located on a helix adjacent to the relay helix. After BSL labeling, myosin was bound to actin in oriented muscle fibers, making it possible to measure helix orientation relative to the fiber axis. Spectra were acquired in APO and ADP states. Simulation of the 494.498.BSL spectra demonstrates that in APO and ADP states there are two highly ordered populations of the relay helix. APO and ADP spectra contain the same two spectral components, but the distribution of these two components differs between states. Similarly, spectra from the 639.643 construct reveal an ADP-induced structural transition, but the difference between APO and ADP spectra demonstrates a 3o rotation of the 639.643 helix. Our results demonstrate structural transitions of two helixes within the CD of actin-bound myosin associated with nucleotide binding. Measuring these transitions is essential to understanding the molecular mechanism of force generation. This is particularly true for the relay helix, which plays a key role in the coupling of myosin ATPase and motor function. Additionally, these results demonstrate the utility of BSL for measuring transitions in protein orientation, order, and dynamics.

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

We are using electron paramagnetic resonance and site-directed spin labeling to measure the orientation of the light chain domain of skeletal muscle myosin in pre-power stroke states trapped by the phosphate analog AlF4. Previous work (Kraft et al. (2005) Proc. Natl. Acad. Sci. USA 102:13861-66) has shown via single-fiber X-ray diffraction that AlF4 traps two distinct pre-powerstroke myosin states in activated muscle. The first state (ADP.AlF4-I) produces a weak actin binding, disordered myosin whereas the second state (ADP.AlF4-II) produces a strong binding, stereospecific actomyosin complex. This weak-to-strong transition is a clear indication of the initiation of the power stroke before phosphate release, but it does not reveal the state of the light chain domain (LCD), which undergoes a rotation during the power stroke. We measured the orientation of the light chain domain by exchanging the native regulatory light chain (RLC) of skinned rabbit psoas muscle fiber bundles with a Di-Cys mutant RLC labeled with a bifunctional spin label. Our group has shown previously (Thompson and Naber et al. 2008 Biophys J, in press) that a bifunctional methanethiosulfonate spin label binds rigidly to myosin and reports protein orientation accurately. EPR spectra of oriented fibers show that the LCD produces a distinct orientation from rigor and relaxation in the ADP.AlF4-II state whereas the ADP.AlF4-I state is indistinguishable from relaxed muscle, supporting the hypothesis that the II state represents a state early in the power stroke with a distinct LCD orientation. This work was supported by NIH (AR32961, AR007612). We thank Bernhard Brenner and Theresia Kraft for guidance.