Effect of nucleotides and pyrophosphate on spin labels bound to S1 thiol groups of myosin

Seidel, John C.; Chopek, M.; Gergely, J.

doi:10.1021/bi00818a021

Cited by 95 publications

(74 citation statements)

References 23 publications

(17 reference statements)

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“…A series of major papers on the metal and nucleotide binding properties of actin followed in the 1960s. His work with John (Jack) Seidel and David Thomas pioneered use of spin-labeling techniques to probe the structure of myosin (Seidel et al 1970;Thomas et al 1975).…”

Section: Scientific Contributions and Professional Honorsmentioning

confidence: 99%

John Gergely (1919–2013): a pillar in the muscle protein field

Greaser

Potter

Thomas

et al. 2013

J Muscle Res Cell Motil

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Section: Scientific Contributions and Professional Honorsmentioning

confidence: 99%

John Gergely (1919–2013): a pillar in the muscle protein field

Greaser

Potter

Thomas

et al. 2013

J Muscle Res Cell Motil

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“…Conformational changes within the myosin head during steady-state ATP hydrolysis have been detected by observing changes in the UV absorbance [53], intrinsic fluorescence [30,31,54], biochemical crosslinking [55,56], electron paramagnetic resonance spectroscopy [57][58][59], electron microscopy [60][61][62], NMR spectroscopy [63], fluorescent labeling [64], fluorescence energy transfer [65], small-angle X-ray scattering [66]. In addition, fluorescence polarization is highly sensitive to conformational changes in myosin [67][68][69].…”

Section: Conformational Studies Of Myosin S1mentioning

confidence: 99%

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Gawalapu¹,

Root²

2006

Archives of Biochemistry and Biophysics

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Gawalapu, Ravi Kumar. Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft. Doctor of Philosophy (Biochemistry), August 2007, 148 pp., 3 tables, 60 illustrations, 120 references, 120 titles.In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy.Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actinactivated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin

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“…The highly reactive SH-1 thiol of myosin was labeled with N-(l-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacctamide as described by Seidel et al [23]. Myosin was reacted with 2 mol spin label/mol myosin at pH 8.0 for 1 h at 4 T .…”

Section: Labeling Qf the Sh-i And Sh-i + Sh-2 Moieties (Spin Labeling)mentioning

confidence: 99%

“…The reaction mixture was then exhaustively dialyzed against 0.5 M KCl, 20 mM Tes, and 1 mM EDTA, pH 7.0 (4°C) to remove unreacted label, as described by Seidel et al [23] and Graceffa and Seidel [24].…”

Section: Labeling Qf the Sh-i And Sh-i + Sh-2 Moieties (Spin Labeling)mentioning

confidence: 99%

See 1 more Smart Citation

Modification of the Rapidly Reacting Thiols of Atrial and Ventricular Myosins

Srivastava

Wikman‐Coffelt

1982

European Journal of Biochemistry

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Environmental variances were noted around the rapidly reacting SH-1 thiol of ventricular versus atrial myosin, based on electron paramagnetic resonance studies. Further studies, in which either SH-1 or SH-1 + SH-2 thiols were modified with N-ethylmaleimide, indicated the importance of the SH-2 moiety of both isozymes for gcneration of tension, when analyzed as synthetic actomyosin threads. Comparative EPR studies showed that the spin label was more strongly immobilized when complexed to ventricular SH-1 thiol as compared to when it was complexed to atrial myosin. Likewise, addition of PPi, ATP or ADP created a greater mobility in the spin label when added to ventricular spin-labeled myosin as compared to that of atrial myosin. Comparative studies of spin-labeled actomyosin versus myosin analyzed at different EPR power settings also demonstrated disparaties surrounding the SH-1 thiol between the two myosin isozymes.The living organism contains several types of myosins which vary with the function of the tissue. Myosin from the fast-contracting skeletal muscle can be distinguished from that of a slow-contracting one by observable differences in the myosin isozymes [I], which are products of distinct genes [2]. Cross-inncrvation of fast-twitch and slow-twitch muscle produces a reciprocal transformation of the physiological responses of the muscle, which appears to be a reprogramming of protein synthesis within the muscles. New isozymes of light chains [3] and myosin heavy chains [4] arc synthesized following cross-reinnervation. Thcre are comparable variances in the heart; myosin from the fast-contracting chambers [5]. the atria, can be distinguished from the more slowly contracting ones, the ventricles, by observable differences in myosin isozymes [6], which include disparities in the primary structure of both the light [7] and heavy chains [8]. The light chains are immunologically distinct between the two tissues [9], however, there is partial identity between the heavy chains of atrial and ventricular myosins [9]. One of the multiple ventricular heavy chains [lo] is homologous to that of atrial myosin heavy chains [Ill. Similar to variances in innervation of fast and slow skeletal muscle, there are anomalies in the innervation between the atria and ventricles. Innervation to the atria allows for both parasympathetic and sympathetic ganglion-stimulating substances, whereas innervation to the ventricles is only sympathomimetic [12,13].Two functionally active sulfhydryls, the rapidly reacting SH-1 thiol [I41 and the more slowly reacting SH-2 thiol [IS] separatcd by only nine amino acids [16], located in the head region of myosin, move as close as 0.2nm together in the presence of MgATP [17]. Although an 8000-dalton CNBr peptide containing the SH-1 and SH-2 thiols is similar between atrial and ventricular bovine myosins [18], masking of Abbreviations. EPR, electron paramagnetic resonance; Tes, 2-{[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino}-ctlian~ulphonic acid.the SH-1 thiol with N-ethylmaleimide causes an a...

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Effect of nucleotides and pyrophosphate on spin labels bound to S1 thiol groups of myosin

Cited by 95 publications

References 23 publications

John Gergely (1919–2013): a pillar in the muscle protein field

John Gergely (1919–2013): a pillar in the muscle protein field

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Modification of the Rapidly Reacting Thiols of Atrial and Ventricular Myosins

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