BACKBONE DYNAMICS OF (1-71)BACTERIOOPSIN AND (1-36)BACTERIOOPSIN STUDIED BY 2-DIMENSIONAL H-1-N-15 NMR-SPECTROSCOPY

The backbone dynamics of uniformly N-15-labelled fragments (residues 1 -71 and 1-36) of bacterioopsin, solubilized in two media (methanol-chl oroform (1:1), 0.1 M (HCO2NH4)-H-2, or SDS micelles) have been investi gated using 2D proton-detected heteronuclear H-1-N-15 NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of th e conformational exchange to the transverse relaxation rates of indivi dual nitrogens were elucidated using an set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high -frequency CPMG spin-lock. We found that most of the backbone amide gr oups of (1-71)bacterioopspin in SDS micelles are involved in the confo rmational exchange process over a rate range of 10(3) to 10(4) s(-1). This conformational exchange is supposed to be due to an interaction b etween two alpha-helixes of (1-71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. N-15 relaxation rates and H-1-N-15 NOE valu es were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A (1982). J Am. Chem. Soc., 104, 4546-4559]. In addition to overall rotation of the molecule, the backbone N-H vector s of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the alpha-helical stretches was mostl y attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro(50), i ndicating an anisotropy of the overall rotational diffusion of the mol ecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the alpha-helixes of (1-71)bac terioopsin. The internal dynamics of the alpha-helixes on the millisec ond and nanosecond time scales should be taken into account in the dev elopment of a model of the functioning bacteriorhodopsin.