Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE.

doi:10.1021/BI00485A023

Paper

Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE.

Published Aug 21, 1990 · K. Ajtai, L. Pótó, T. Burghardt

Biochemistry

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13

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2

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Abstract

The nitroxide spin label (iodoacetamido)proxyl (IPSL) was specifically and rigidly attached to sulfhydryl 1 (SH1) on myosin subfragment 1 (S1). The specificity of this label for SH1 was demonstrated by using a technique where the spin label is localized on the electrophoresis-isolated proteolytic fragments of myosin using electron paramagnetic resonance (EPR). Studies of the rigidity of the probe on SH1 indicate that the IPSL is immobilized on the surface of S1 in the presence and absence of the nucleotides MgADP or MgATP. The EPR spectrum of muscle fibers decorated with IPSL-S1 shows that the IPSL-S1 rotates from its orientation in rigor upon binding MgADP. The angular displacement due to nucleotide binding is larger than that detected with the (maleimido)tempo spin label [Ajtai, K., French, A. R., & Burghardt, T. P. (1989) Biophys. J. 56, 535-541], demonstrating that the IPSL is oriented on the myosin cross-bridge in a manner that is favorable for detecting cross-bridge rotation during the rigor to MgADP state transition.

In Vitro Study

Study Snapshot

The (iodoacetamido)proxyl spin-labeled myosin subfragment 1 (IPSL-S1) is specific for sulfhydryl 1 on myosin subfragment 1, and its orientation on the myosin cross-bridge allows for detection of

PopulationOlder adults (50-71 years)

Sample size24

MethodsObservational

OutcomesBody Mass Index projections

ResultsSocial networks mitigate obesity in older groups.

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Paper

Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE.

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References

Citations

Orientation Distribution of Molecules: Characterization and Experimental Determination by Means of Magnetic Resonance

Magnetic resonance is the most informative technique for detailed characterization of molecular orientation distribution in partially aligned media.

Paramagnetic NMR in drug discovery

Paramagnetic NMR provides valuable structural restraints for drug discovery, particularly in early stages when crystallography is intractable, aiding in filtering computational docking poses and guiding medicinal chemistry.

Inhibition of myosin ATPase by metal fluoride complexes.

Magnesium and MgADP form a complex that traps the active site of myosin subfragment 1 and inhibits myosin ATPase, with free fluoride necessary for maintaining a stable trap and suggesting looser binding to the bridging oxygen.

Probes bound to myosin Cys-707 rotate during length transients in contraction.

Myosin cross-bridges rotate, causing muscle contraction by impelling actin filaments, and changes in rhodamine probe attitude correlate with tension changes in contraction.

Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.

The conformations of S1.ADP.AlF4-, S1.ADP.VO(4)3-, and S1.ADP.BeFx complexes in the Cys707-Cys697 region of myosin subfragment 1 are distinct, with the latter two partially res