Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine

Biophysical Journal
C Sabido-DavidMalcolm Irving

Abstract

The regulatory light chain (RLC) from chicken gizzard myosin was covalently modified on cysteine 108 with either the 5- or 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLCs were purified by fast protein liquid chromatography and characterized by reverse-phase high-performance liquid chromatography (HPLC), tryptic digestion, and electrospray mass spectrometry. Labeled RLCs were exchanged into the native myosin heads of single skinned fibers from rabbit psoas muscle, and the ATR dipole orientations were determined by fluorescence polarization. The 5- and 6-ATR dipoles had distinct orientations, and model orientational distributions suggest that they are more than 20 degrees apart in rigor. In the rigor-to-relaxed transition (sarcomere length 2.4 microm, 10 degrees C), the 5-ATR dipole became more perpendicular to the fiber axis, but the 6-ATR dipole became more parallel. This orientation change was absent at sarcomere length 4.0 microm, where overlap between myosin and actin filaments is abolished. When the temperature of relaxed fibers was raised to 30 degrees C, the 6-ATR dipoles became more parallel to the fiber axis and less ordered; when ionic strength was lowered from 160 mM to 20 mM (5 degrees C), the 6-AT...Continue Reading

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Citations

Jun 27, 2002·Journal of Molecular Biology·Seth C HopkinsYale E Goldman
Nov 15, 2006·Proceedings of the National Academy of Sciences of the United States of America·Yin-Biao SunMalcolm Irving
Apr 14, 2010·Proceedings of the National Academy of Sciences of the United States of America·V NucciottiF S Pavone
Sep 12, 2013·Biochimica Et Biophysica Acta·Stephanie B WallAimee Landar
Apr 2, 2003·Biophysical Journal·J Borejdo, I Akopova
Jul 16, 2008·Biophysical Journal·Andrea C KnowlesMalcolm Irving
Mar 26, 2004·Biophysical Journal·J BorejdoT P Burghardt
Feb 16, 2005·Journal of Muscle Research and Cell Motility·A A ShepardJ Borejdo
Sep 13, 2002·American Journal of Physiology. Cell Physiology·Sophie B P ChargéSimon M Hughes
Jun 6, 2006·American Journal of Physiology. Heart and Circulatory Physiology·D DumkaJ Borejdo
Dec 29, 2005·Experimental Biology and Medicine·Julian BorejdoIrina Akopova
Mar 15, 2015·American Journal of Physiology. Heart and Circulatory Physiology·D DuggalJ Borejdo
Dec 1, 2012·Biophysics Reviews·J BorejdoK Midde
Feb 14, 2017·Methods and Applications in Fluorescence·Deborah Y ShroderYale E Goldman
Apr 30, 2003·Molecular Cell·Roisean E FergusonMalcolm Irving

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