title: NMR studies of the dynamics of a bifunctional rhodamine probe attached to troponin C.
abstract: Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton T6G 2H7, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Julien
authors.forename: Pascal
authors.initials: P
authors.lastname: Mercier
authors.forename: Leo
authors.initials: L
authors.lastname: Spyracopoulos
authors.forename: John E T
authors.initials: JE
authors.lastname: Corrie
authors.forename: Brian D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D008958:Models, Molecular; D015394:Molecular Structure; D012015:Reference Standards; D012235:Rhodamines / Q000737:chemistry*; D019209:Troponin C / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D019209:Troponin C