Explicit Treatment of Spin Labels in Modeling of Distance Constraints from Dipolar EPR and DEER

Sale, Ken; Song, Likai; Liu, Yi-Shiuan; Perozo, Eduardo; Fajer, Piotr G.

doi:10.1021/ja051652w

Cited by 117 publications

(117 citation statements)

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“…S2), they serve to clarify the interpretation of DEER distance measurements (Fig. 7) (22). When backbone carbon atoms were restrained, the distance distributions were narrower than the experimental ones, but relaxation of restraints gave broader distributions, with widths closer to the observed ones.…”

Section: Resultsmentioning

confidence: 78%

“…1) were modified to include missing loops and residues by using InsightII, and native residues were mutated to spin-labeled Cys by using Visual Molecular Dynamics (VMD) (43). Parameters for IPSL and MSL were based on CHARMM19 united-atom force fields (22). Metropolis Monte Carlo minimization (22,44) was used to determine starting points for MD simulations.…”

Section: Methodsmentioning

confidence: 99%

“…Parameters for IPSL and MSL were based on CHARMM19 united-atom force fields (22). Metropolis Monte Carlo minimization (22,44) was used to determine starting points for MD simulations. MD simulations were carried out essentially as described previously (21, 25) (see also SI Text).…”

Section: Methodsmentioning

confidence: 99%

“…Twentieth-century EPR methods, based on continuous wave (CW) spectroscopy, were sensitive only to distances in the range of 0.7-2.5 nm (19), but now the pulsed electron double resonance technique called double electron-electron resonance (DEER) is sensitive in the range of 1.7-6 nm (20). Computational simulations of protein dynamics, explicitly including the bound spin label (21), are effective in increasing the reliability of EPR data analysis, especially in the case of DEER (22). To examine cleft closure in myosin, we have engineered cysteine spin labeling sites in a Dicty myosin II construct lacking other reactive Cys.…”

Section: Figmentioning

confidence: 99%

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Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Klein

Burr

Svensson

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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We present a structurally dynamic model for nucleotide-and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solventfilled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.double electron-electron resonance ͉ dipolar interaction ͉ actomyosin M yosin motors use the energy derived from ATP hydrolysis to generate force for a diverse repertoire of biological tasks (1). Class II myosins produce muscle contraction and are involved in cytokinesis and cell motility. The present study focuses on Dictyostelium discoideum (Dicty) myosin II, which offers optimal facility for expression and purification of mutants, and is functionally similar to muscle myosin II (2, 3).

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Section: Resultsmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Klein

Burr

Svensson

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…This results in flexibility of the spin label, and uncertainty of its position due to rotamer diversity. To evaluate whether the wide distance distributions were due solely to rotamer diversity and spin-label flexibility, we performed molecular modeling using Metropolis Monte Carlo minimization (MMCM) (15,16), which can find the lowest energy rotamer (i.e., the most probable conformation) within the constraints of a local structure. Using the coordinates from the Wendt model, the most probable rotamer conformations for MTSSL on the RLC were found for each of the labeled sites (Table S2 and Fig.…”

Section: Resultsmentioning

confidence: 99%

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Vileno

Chamoun

Liang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the shorttailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the offstate, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.computational modeling | structural biology M yosin II is a motor protein that can transduce chemical energy from ATP hydrolysis into mechanical work. This process involves cyclic interactions between actin in thin and myosin in thick filaments causing relative filament sliding. Smooth muscle myosin (SMM), which contains two heads, each with a motor domain (MD) and regulatory domain (RD), connected by a long coiled-coil tail domain is an example of a large multisubunit protein that is allosterically regulated by phosphorylation (for review, see ref. 1). Phosphorylation of a single serine on each regulatory light chain (RLC), which are about 100 Å distant from the active sites, is sufficient to transform the protein from a state in which the ATPase activity is weakly actin-activated to one in which it is strongly actin-activated by controlling the rate of Pi release (2). Because phosphorylation modulates the rate of ADP release (3) and also alters the attitude of the lever arm domain with respect to the MD (4), it is likely that phosphorylation modulates strain between the two heads (5).The structural basis for the allosteric regulation has been a topic of intense study. It is a difficult problem because regulation by phosphorylation requires the presence of both head domains. Cryogenic EM studies of double-headed constructs (6-9) have incorporated data fr...

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Site‐directed Spin Labeling and Pulse Dipolar Electron Paramagnetic Resonance

Klare

Steinhoff

2010

Encyclopedia of Analytical Chemistry

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Structural and functional investigations on biomolecules often rely on the availability of topological information, either to allow building structural models or to characterize conformational changes of these molecules or complexes thereof during function. Site‐directed spin labeling (SDSL) in combination with EPR spectroscopy became a very powerful tool to obtain intra‐ and inter‐molecular distance constraints, as the accessible distances span the interesting range from 0.5–8 nm, crystallization is not required, and the size of the system under investigation is not limited. Especially the development of pulse dipolar EPR methods, in particular double electron–electron resonance (DEER) spectroscopy with its ability to provide interspin distance distributions in the 2–8 nm range, greatly increased the applicability of electron paramagnetic resonance (EPR) spectroscopy for studies on biological systems. This article is intended to give an introduction into SDSL and dipolar EPR. The first part summarizes the state‐of‐the‐art in SDSL, providing an overview of methodologies currently available or under development. In the second part, the technique of DEER spectroscopy is introduced, covering the basic theoretical background as well as selected practical aspects of data acquisition and analysis. Finally, selected examples for the application of SDSL‐DEER from the recent literature are summarized.

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Explicit Treatment of Spin Labels in Modeling of Distance Constraints from Dipolar EPR and DEER

Cited by 117 publications

References 11 publications

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Site‐directed Spin Labeling and Pulse Dipolar Electron Paramagnetic Resonance

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