A high-resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme. Dynamic behavior of protein and detergent in the complex.
We have recorded 100.6-MHz high-resolution solid-state 13C-NMR spectra of crystalline cytochrome-c oxidase from bovine heart muscle and hen egg-white lysozyme, to compare conformation and dynamics of a typical membrane-protein complex with those of lysozyme. The absence of severe interference with the solid-state 13C-NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C-NMR feature of cytochrome-c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively. The dynamic behavior of the backbone and side chains of cytochrome-c oxidase was compared with that of lysozyme on the basis of the 13C spin-lattice relaxation times (T1): the backbone of the cytochrome-c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin-lattice relaxation time in the rotating frame (TH1 rho) and cross-polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.
Tuzi
S
S
Department of Life Science, Himeji Institute of Technology, Hyogo, Japan.
Shinzawa-Itoh
K
K
Erata
T
T
Naito
A
A
Yoshikawa
S
S
Saitô
H
H
D003461:Crystallography; D003902:Detergents / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009038:Motion; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D014961:X-Ray Diffraction
D003902:Detergents; D003576:Electron Transport Complex IV; D009113:Muramidase