Probing cross-bridge angular transitions using multiple extrinsic reporter groups.
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 degrees 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 rotation motion of cross-bridges during force generation.
Ajtai
K
K
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Ringler
A
A
Burghardt
T P
TP
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008958:Models, Molecular; D009132:Muscles / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry*; D011817:Rabbits; D012996:Solutions; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D012996:Solutions; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl