Structure and Dynamics of the Force-Generating Domain of Myosin Probed by Multifrequency Electron Paramagnetic Resonance

Nesmelov, Yuri E.; Agafonov, Roman V.; Burr, Adam R.; Weber, Ralph T.; Thomas, David D.

doi:10.1529/biophysj.107.124305

Cited by 27 publications

(34 citation statements)

References 42 publications

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“…The coexistence of two structural states (M Ã and M ÃÃ ) in a single biochemical state (the myosin-ATP complex), during the transient phase of the myosin ATPase reaction, confirms our previous findings with nucleotide analogs bound to myosin at equilibrium (11,21), implying loose coupling between myosin structural and biochemical states. This conclusion is further supported by the considerable width of the detected distance distributions (Fig.…”

Section: Discussionsupporting

confidence: 89%

“…For example, in the M Ã crystal structure, switch loop I and switch loop II of the active site do not appear to be in the proper position for catalysis of ATP hydrolysis (14,16), suggesting that step 3′ in Scheme 3 is not likely to occur. Indeed the present study, coupled with previous studies, indicates precisely that structural changes in the force-generating region (11,21) and the actin-binding cleft (22) are not tightly coupled to ligand binding at the active site. Rather, each crystal structure represents a trapped structural state among the much larger repertoire available to each biochemical state in solution (4).…”

Section: Discussionsupporting

confidence: 80%

“…Change in the intrinsic fluorescence of a myosin-nucleotide analog complex upon pressure and temperature jumps suggests a transition between two structural states of myosin (17). Spectroscopic data on myosin with probes at the active site (18,19), in the forcegenerating region (11,20,21), or in the actin-binding cleft (22) show evidence for two resolved structural states (conformations) in the presence of a single biochemical state (bound ligand). TR-FRET has provided a clear structural picture of this phenomenon: With a single nucleotide analog bound to myosin, two distinct structural states (M Ã straight and M ÃÃ bent) were resolved for the entire head (23) (Fig.…”

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Structural kinetics of myosin by transient time-resolved FRET

Nesmelov

Agafonov

Negrashov

et al. 2011

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

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For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, ðTRÞ 2 FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. ðTRÞ 2 FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond timeresolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by ðTRÞ 2 FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.disorder-to-order transition | myosin II | dictyostelium S tructural dynamics lies at the heart of protein function. Transitions among distinct structural states, each characterized by both structural order and internal dynamic disorder, are typically required for the function of a protein, especially an enzyme. To describe the mechanism of a protein's function is to describe its structural kinetics, i.e., the coupling of protein structural transitions to biochemical kinetics, as defined by changes in bound ligand (1-3). However, for most proteins there remains mechanistic ambiguity due to a gap between structural data, determined primarily from static protein crystals, and kinetics, measured during the transient phase of the biochemical reaction. In the present study, we have closed this gap by measuring structure and kinetics simultaneously, using myosin as a powerful example.In a molecular motor, ATP binding and hydrolysis initiate protein structural changes that lead to force generation, and characterization of motor protein structural kinetics is essential to understand the protein in action. Myosin is a molecular motor responsible for actin-dependent force generation and movement in muscle and nonmuscle cells; it works cyclically, producing mechanical work on actin using energy from ATP hydrolysis (recently reviewed in refs. 4 and 5). Transient kinetics in myosin has been typically monitored by tryptophan fluorescence (6, 7), but that signal provides no direct structural information. FRET does provide structural information, in the form of interprobe distance, and transient FRET experiments have provided a glimpse of structural kinetics in muscle and other protein...

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 80%

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confidence: 99%

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Structural kinetics of myosin by transient time-resolved FRET

Nesmelov

Agafonov

Negrashov

et al. 2011

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

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“…5), suggesting that the inner cleft is more directly coupled to switch II, which differs in all three structural states (7). Similarly, in both rabbit (33) and Dicty (34), a spin label in the force-generating region of myosin resolves three distinct structural states that change their populations with biochemical state. Continued analysis of the complex structural and biochemical coupling among the subdomains of myosin and actin, in space and time, is needed to understand the mechanism of actomyosin function.…”

Section: Sdsl-epr Methodology and Computationalmentioning

confidence: 99%

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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“…These structural states are assumed to be tightly coupled to biochemical states, which are defined by the nucleotide bound to the active site. These conformational states of myosin S1 have also been observed and characterized by EPR through the changes in mobility of a nitroxide spin probe (IASL) at the SH1 labeling site (C707) Barnett and Thomas 1987;Nesmelov et al 2008;Ostap et al 1993;Seidel et al 1970) in the force-generating region of myosin.…”

Section: Myosin S1-iasl Dynamics In Solutionmentioning

confidence: 99%

Protein Structural Dynamics Revealed by Site-Directed Spin Labeling and Multifrequency EPR

Nesmelov¹

2013

Protein Dynamics

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Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.

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Structure and Dynamics of the Force-Generating Domain of Myosin Probed by Multifrequency Electron Paramagnetic Resonance

Cited by 27 publications

References 42 publications

Structural kinetics of myosin by transient time-resolved FRET

Structural kinetics of myosin by transient time-resolved FRET

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

Protein Structural Dynamics Revealed by Site-Directed Spin Labeling and Multifrequency EPR

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