Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR |
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Authors: | Izuru Kawamura Junko Tanabe Hazime Saitô |
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Institution: | a Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan b Graduate School of Science, University of Hyogo, Harima Science Garden City, Kamigori, Hyogo 678-1297, Japan c Center for Quantum Life Sciences, Hiroshima University, Higashi-Hiroshima, Japan |
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Abstract: | 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 T1ρ) 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 β-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 T1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal. |
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Keywords: | bR bacteriorhodopsin bO bacterio-opsin PM purple membrane CP-MAS Cross polarization-magic angle spinning REDOR Rotational echo double resonance |
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