Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics

Roopnarine, Osha; Hideg, Kálmán; Thomas, David D.

doi:10.1016/s0006-3495(93)81561-3

Cited by 21 publications

(55 citation statements)

References 37 publications

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“…However, complementary experiments with SHi-bound maleimide spin labels (MSL), indane-dione spin labels (InVSL), or phosphorescent dyes, which bind rigidly to myosin heads even in the presence of ATP and report the global behavior of myosin heads, can be compared directly with this study. In agreement with the present study on IASL-fibers, the EPR spectrum of SHlbound MSL (Thomas and Cooke, 1980;Fajer et al, 1990a,b) or InVSL (Roopnarine et al, 1993;Roopnarine solved. As the apparent equilibrium constant for ATP hy- Intermediates and step numbers are defined as in Scheme 1, except that ATP and ADP are abbreviated as T and D, respectively.…”

Section: Relationship To Other Studiessupporting

confidence: 93%

“…This leaves open the possibility that some heads contain additional spin labels attached to sites that do not affect the ATPase activity. However, the number of spin labels bound per myosin head (determined as described by Roopnarine et al, 1993) was found to be 0.68 ± 0.08, precisely matching the fraction of heads with inhibited K/EDTA-ATPase, implying that virtually all of the spin labels are bound to SHi, to the exclusion of all other sites in the fiber (Matta and Thomas, 1992;Matta, 1994). Fig.…”

Section: Specificity Of Spin Labelingmentioning

confidence: 85%

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Resolution of three structural states of spin-labeled myosin in contracting muscle

Ostap

Barnett

Thomas

1995

Biophysical Journal

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We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.

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Section: Relationship To Other Studiessupporting

confidence: 93%

Section: Specificity Of Spin Labelingmentioning

confidence: 85%

Resolution of three structural states of spin-labeled myosin in contracting muscle

Ostap

Barnett

Thomas

1995

Biophysical Journal

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“…Thus, the InVSL label is immobilized both on the nanosecond time scale of EPR and on the millisecond time scale of ST-EPR experiments, and therefore can be used to study the dynamics of the regulatory domain. The InVSL was previously shown to be rigidly attached to Cys-707 of the catalytic domain using the same criteria (18). Mobility in Myosin Filaments.…”

Section: Resultsmentioning

confidence: 99%

Independent mobility of catalytic and regulatory domains of myosin heads

Adhikari

Hideg

Fajer

1997

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

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The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer-EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time ( R ) between 24 and 48 s. In contrast, the mobility of the catalytic domain was found to be R ‫؍‬ 5-9 s. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with R ‫؍‬ 1-4 s and >1,000 s, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spinlabeled sites. The most straightforward interpretation suggests a f lexible hinge between the two domains, which would have to stiffen before force could be generated.The myosin head (subfragment 1, S1) plays a central role during muscle contraction. It is thought that during the ATPase cycle S1 interacts with actin and undergoes a series of specific changes in its conformation, resulting in a directional strain on the actin filaments. The nature of these conformational changes and the mechanism by which they produce the directed force is still unclear (1, 2), although the original model of Huxley and Simmons (3) provides a valid framework. The determination of the atomic structures of S1, actin, and the low-resolution structure of the acto-S1 complex has led to a hypothesis of a structural model, in which helix movement is generated by the hydrolysis of ATP and the formation of a stereospecific acto-S1 complex generates torque within the catalytic domain, which is then amplified by the regulatory domain acting as a lever arm (4, 5). Rayment's model was preceded by the observation of shape changes in the myosin heads between various chemical states of myosin. A decrease in the hydrodynamic size of S1 upon MgADP binding and upon ATP hydrolysis was observed by Highsmith and Eden (6) and by Wakabayashi et al. (7,8). More recently, low-resolution electron microscopy reconstitution of S1-decorated actin filaments and EPR studies have indicated a rotation of the regulatory domain with respect to the catalytic domain (9-11). In these cases, the rotation was observed at the distal end of the regulatory domain on th...

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“…Myofibrils were prepared from fiber bundles and assayed for protein concentration as described by Roopnarine and Thomas (1994b). High-salt (K/EDTAand Ca/K-) ATPase assays were performed on myofibrils as described by Roopnarine et al (1993). The fraction of heads labeled at SHI and/or SH2 (fsH) was determined to be 0.63 ± 0.05 as described in Roopnarine and Thomas (1994b), from the fractional inhibition of the K/EDTA-ATPase activity of the labeled sample (KL) relative to the unlabeled controls (KUL):fsH = (KUL -KL)/(KUL -K.), where K., the activity of maximally labeled myosin heads in myofibrils, was determined to be 0.04 ,imol Pi/mg/min (Roopnarine and Thomas, 1994b).…”

Section: Preparations and Assaysmentioning

confidence: 99%

“…We have recently shown that this need is met by a newly synthesized indane dione spin label, designated InVSL (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]indane-1,3-dione) ( Roopnarine and Thomas, 1994b). InVSL is covalently and rigidly immobilized on myosin, so that it reports the global rotation of myosin heads (Roopnarine et al, 1993), and it binds to muscle fibers in rigor with its principal axis almost precisely parallel to the fiber axis, making its EPR spectra directly and unambiguously sensitive to axial rotations of the myosin heads (Roopnarine and Thomas, 1994b). Therefore, in the present study, we have used conventional EPR spectroscopy of InVSL-labeled muscle fibers to study the orientation of myosin heads in rigor, relaxation, and isometric contraction.…”

Section: Introductionmentioning

confidence: 99%

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers

Roopnarine¹,

Thomas²

1995

Biophysical Journal

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We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.

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Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics

Cited by 21 publications

References 37 publications

Resolution of three structural states of spin-labeled myosin in contracting muscle

Resolution of three structural states of spin-labeled myosin in contracting muscle

Independent mobility of catalytic and regulatory domains of myosin heads

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers

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