Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR.
Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.
Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
Izuru
I
Kawamura
Masato
M
Ohmine
Junko
J
Tanabe
Satoru
S
Tuzi
Hazime
H
Saitô
Akira
A
Naito
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry; D019617:Halobacterium salinarum / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D017433:Protein Structure, Secondary; D011522:Protons
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011522:Protons; D001436:Bacteriorhodopsins