Specificity and Orientation of (Iodoacetamido)proxyl Spin-Labeled Myosin Subfragment 1 Decorating Muscle Fibers: Localization of Protein-Bound Spin Labels Using SDS-PAGE

Katalin Ajtai, László Pótó, Thomas P. Burghardt

Research output: Contribution to journalArticle

10 Scopus citations

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 Mg ATP. 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.

Original languageEnglish (US)
Pages (from-to)7733-7741
Number of pages9
JournalBiochemistry
Volume29
Issue number33
DOIs
StatePublished - Aug 1 1990

ASJC Scopus subject areas

  • Biochemistry

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