Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.
Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-(13)C], [1,4-(13)C] and [2,3-(13)C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual (13)C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-(13)C]-succinic acid or [2,3-(13)C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-(13)C]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3 (13)C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures.
van Gammeren
A J
AJ
Leiden Institute of Chemistry, Gorlaeus Laboratoria, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
Hulsbergen
F B
FB
Hollander
J G
JG
de Groot
H J M
HJ
D000596:Amino Acids / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry*; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012241:Rhodopseudomonas / Q000737:chemistry*; D013113:Spin Labels; D019802:Succinic Acid / Q000737:chemistry
D000596:Amino Acids; D002247:Carbon Isotopes; D045342:Light-Harvesting Protein Complexes; D013113:Spin Labels; D019802:Succinic Acid