Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Biochim Biophys Acta. 2007 Dec;1768(12):3090-7. doi: 10.1016/j.bbamem.2007.11.001. Epub 2007 Nov 12.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteriorhodopsins / chemistry*
  • Carbon Isotopes / chemistry
  • Halobacterium salinarum / metabolism
  • Magnetic Resonance Spectroscopy / methods*
  • Molecular Sequence Data
  • Nitrogen Isotopes / chemistry
  • Protein Structure, Secondary
  • Protons

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes
  • Protons
  • Bacteriorhodopsins