Structural Dynamics of the Actomyosin Complex Probed by a Bifunctional Spin Label that Cross-Links SH1 and SH2

Thompson, Andrew R.; Naber, Nariman; Wilson, Clyde F.; Cooke, Roger; Thomas, David D.

doi:10.1529/biophysj.108.138982

Cited by 24 publications

(48 citation statements)

References 47 publications

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“…M ÃÃ :T is the state with maximum entropy in this process (steps 1-3, Scheme 2), reflecting high configurational disorder, and the subsequent transition state 3 has the lowest entropy and highest free energy. Thus a disorderto-order transition provides the driving force for the subsequent power stroke, which is consistent with a recently trapped actomyosin intermediate (30), suggesting that a disorder-to-order transition is the first step in force generation (30)(31)(32).…”

Section: Resultssupporting

confidence: 80%

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: Resultssupporting

confidence: 80%

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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“…EPR spectra were recorded at X-band (9.6 GHz) using an E500 EleXsys spectrometer (Bruker Instruments). Acquisition of spectra for oriented samples was performed as described previously (21,22). BSL spectra Values are derived using probe orientations obtained either from EPR (Experimental columns) or directly from a cryo-EM model of actomyosin (Model-based column) (32).…”

Section: Methodsmentioning

confidence: 99%

“…1A). This bifunctional spin label (BSL) is rigidly immobilized when reacted with a pair of Cys residues, so that it undergoes negligible ns rotational motion relative to the protein and can thus be used reliably to measure μs protein rotational motions by saturation transfer EPR (21)(22)(23).…”

mentioning

confidence: 99%

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Binder

Cornea

Thompson

et al. 2015

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

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Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1°accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2°accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.T he myosin family of molecular motors is responsible for numerous vital functions in eukaryotes, including the contraction of striated muscle. Bundled within an intricate and highly regulated myofibril lattice, muscle myosin II converts the chemical energy released by ATP binding and hydrolysis into mechanical work, executing a series of structural transitions that generate force on actin and shorten each muscle cell (1, 2). Coupling of actin binding, nucleotide hydrolysis, and lever arm movement within myosin's catalytic domain (CD) is essential for proper function of the contractile apparatus (3, 4).Myosin function requires actin, and thus an understanding of its mechanism requires analysis of both proteins in complex. However, no crystals of actin-myosin complexes have been reported, so the resolution of actin-bound myosin structures is currently limited to that of electron microscopy. Furthermore, X-ray crystallography and electron microscopy produce only static structures in frozen or crystalline environments, which cannot accurately render the dynamics, disorder, and structural transitions that are essential to understanding function and pathology (4, 5).In contrast, site-directed spectroscopy can be used to examine the actin-myosin complex under more physiological conditions. Both fluorescence and electron paramagnetic resonance (EPR) have been used in complement to examine the structural dynamics of myosin bound to actin (6, 7). EPR offers superior orientational resolution, due to the high sensitivity of the EPR spectrum to alignment of a spin label in the applied magnetic field. A wellplaced spin label can provide direct information about orientation and dynamics in the vicinity of the labeling site, a strategy that has proven powerful in the study of myosin in oriented muscle fibers (7-9). However, conventional methods for site-direc...

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“…Oriented EPR samples were prepared as previously described (28,29). S1dC-decorated muscle fiber bundles were blotted to remove excess buffer, cut into 0.5 cm lengths, and aligned perpendicular to the long axis of a quartz tissue flat cell; the fiber axis was manually oriented parallel or perpendicular to the external magnetic field.…”

Section: Methodsmentioning

confidence: 99%

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

Moen

Thomas

Klein

2013

Journal of Biological Chemistry

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Background:The actin-binding cleft of myosin is proposed to close upon force generation. Results: Crosslinking the cleft with a bifunctional spin-label weakens binding, eliminates actin activation, and disorders the actomyosin interface. Conclusion: Restricting the cleft traps an actomyosin state with structural dynamics intermediate between strongly and weakly bound. Significance: We have determined the structural dynamics of an elusive intermediate at the threshold of force generation.

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Structural Dynamics of the Actomyosin Complex Probed by a Bifunctional Spin Label that Cross-Links SH1 and SH2

Cited by 24 publications

References 47 publications

Structural kinetics of myosin by transient time-resolved FRET

Structural kinetics of myosin by transient time-resolved FRET

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

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