Probing Cross-Bridge Angular Transitions Using Multiple Extrinsic Reporter Groups

Katalin Ajtai, Andras Ringler, Thomas P. Burghardt

Research output: Contribution to journalArticle

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Abstract

15N-and 2H-substituted maleimido-TEMPO spin label ([15N, 2H]MTSL) and the fluorescent label 1, 5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N, 2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1, 5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90° to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N, 2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1, 5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotational motion of cross-bridges during force generation.

Original languageEnglish (US)
Pages (from-to)207-217
Number of pages11
JournalBiochemistry
Volume31
Issue number1
DOIs
StatePublished - Jan 1 1992

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ASJC Scopus subject areas

  • Biochemistry

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