Backbone dynamics of (1-71)- and (1-36)bacterioopsin studied by two-dimensional (1)H-          (15)N NMR spectroscopy.
The backbone dynamics of uniformly (15)N-labelled fragments (residues 1-71 and 1-36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M (2)HCO(2)NH(4), or SDS micelles) have been investigated using 2D proton-detected heteronuclear (1)H-(15)N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a 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 groups of (1-71)bacterioopsin in SDS micelles are involved in the conformational 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 between two α-helixes of (1-71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. (15)N relaxation rates and (1)H-(15)N NOE values 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 vectors 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 α-helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro(50), indicating an anisotropy of the overall rotational diffusion of the molecule. 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 α-helixes of (1-71)bacterioopsin. The internal dynamics of the α-helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya 16/10, 117871, Moscow, Russia.
V Y
VY
Orekhov
K V
KV
Pervushin
D M
DM
Korzhnev
A S
AS
Arseniev