title: Following the rotational trajectory of the principal hydrodynamic frame of a protein using multiple probes.
abstract: A generalized set of fluorescence polarization intensities and electron paramagnetic resonance (EPR) spectra from multiple fluorescent and EPR probes of a specific site on an oriented and immobilized protein element in a biological assembly, combined with anisotropic rotational relaxation studies of the purified and labeled protein element freely rotationally diffusing in solution, are used to determine the angular distribution of the principal hydrodynamic frame of the protein element in the biological assembly. This multiprobe analysis method removes all of the basic ambiguities in the measured principal frame angular distribution introduced by limitations or symmetries intrinsic to standard fluorescence polarization intensity ratios and EPR spectra. The angular distribution of the principal frame is also more highly resolved than that previously reported for multiprobe determinations of probe angular distributions and is more useful for determining biological mechanisms because it indicates the order and orientation of the protein element rather than that of the extrinsic probe. Application of this method to the determination of the principal hydrodynamic frame distribution of myosin cross-bridges in muscle fibers in four physiological states, including the active isometric state, demonstrates the method's practicality by indicating the path of cross-bridge orientation changes during the active cycle [Ajtai, Toft, & Burghardt (1994) Biochemistry (following paper in this issue)].
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
authors.forename: T P
authors.initials: TP
authors.lastname: Burghardt
authors.forename: K
authors.initials: K
authors.lastname: Ajtai
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008956:Models, Chemical; D009218:Myosins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D009218:Myosins