Residual backbone and side-chain 13C and 15N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy.
This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion (13)C-(13)C correlation experiments with mixing times of 20 and 500 ms and band selective (13)C-(15)N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long alpha-helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the (13)C-(13)C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.
Gammeren
A J van
AJ
Leiden Institute of Chemistry, Gorlaeus Laboratoria, Leiden University, The Netherlands.
Hulsbergen
F B
FB
Hollander
J G
JG
Groot
H J M de
HJ
D000595:Amino Acid Sequence; D025541:Bacteriochlorophyll A / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes; D004058:Diffusion; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry; D009587:Nitrogen Isotopes; D010788:Photosynthesis; D011522:Protons; D012241:Rhodopseudomonas / Q000378:metabolism
D025541:Bacteriochlorophyll A; D002247:Carbon Isotopes; D045342:Light-Harvesting Protein Complexes; D009587:Nitrogen Isotopes; D011522:Protons; D002244:Carbon; D009584:Nitrogen