Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H +-ATP synthase subunit c, by magic-angle spinning solid-state NMR.
Signal assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C-13C and 13C-15N chemical shift correlations, 78% of Calpha, 72% of Cbeta, 62% of C' and 61% of NH signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the 13C-13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by alpha-helices with a loop structure in the middle (at sequence 41-45) as in the case of the solution structure in spite of additional extended conformations at 76-79 at the C-terminus.
Kobayashi
Masatoshi
M
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, 565-0871, Japan.
Matsuki
Yoh
Y
Yumen
Ikuko
I
Fujiwara
Toshimichi
T
Akutsu
Hideo
H
D000595:Amino Acid Sequence; D002247:Carbon Isotopes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry*; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002247:Carbon Isotopes; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D006180:Proton-Translocating ATPases