Backbone dynamics of (1-71)bacterioopsin studied by two-dimensional 1H-15N NMR spectroscopy.

The backbone dynamics of a uniformly 15N-labelled proteolytic fragment (residues 1-71) of bacteriorhodopsin, solubilized in two media [methanol/chloroform (1:1), 0.1 M 2HCO2NH4 and SDS micelles] have been investigated using two-dimensional proton-detected heteronuclear 1H-15N NMR spectroscopy. A set of longitudinal and transverse relaxation rates of 15N nuclei and 1H-15N NOE were obtained for 61 backbone amide groups. The contribution of the conformational exchange to transverse relaxation rates of individual nitrogens was elucidated using a set of different rates of the Carr-Purcell-Meiboom-Gill (CPMG) spin-lock pulse train. We found that most of the backbone amide groups are involved in the co-operative exchange process over the rate range 10(3)-10(4) s-1, with the chemical-shift dispersion near 1 ppm. Contributions of conformational exchange to the measured transverse relaxation were essentially suppressed by the 3-kHz (spin-echo period tau = 0.083 ms) CPMG spin-lock. Under these conditions, the measured longitudinal, transverse relaxation rates and NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. In both media used, the protein exhibits very similar dynamic properties, and has overall rotational correlation times of 7.0 ns and 6.6 ns in organic mixture and in SDS micelles, respectively. In addition to overall rotation of the molecule, the backbone N-H vectors are involved in two types of internal motions; fast, on a time scale of < 20 ps, and intermediate, close to 1 ns. Distinctly mobile regions are identified by a large decrease in the overall order parameter and correspond to N-terminal residues (residues 1-7 both for organic solvent and micelles), C-terminal residues (residues 65-71 and 69-71 for organic solvent and micelles, respectively) and residues connecting alpha helices (residues 33-41 and 33-38, for organic solvent and micelles, respectively). A decrease in the order parameter was also observed for residues next to Pro50, indicating a higher flexibility in this region. Thus, backbone dynamic parameters of (1-71)bacterioopsin are in good correspondence with its spatial structure [Pervushin, K. V., Orekhov, V. Yu., Popov, A., Musina, L. Yu., Arseniev, A. S., (1994) Eur. J. Biochem., in the press]. The observed conformational exchange behavior of alpha helices seems to be induced by the flickering helix-helix interaction and could be important for the functioning of bacteriorhodopsin.