NMR studies of the dynamics of a bifunctional rhodamine probe attached to troponin C

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 1, 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 (13)C-labeled methyl groups on the BR probe and (15)N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance (13)C-R(1 rho), (13)C-R(1) and {(1)H}-(13)C 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 (13)C-(14)N bond with an associated S(s)(2) of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S(2) approximate to 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.