Abstract
Partly biosynthetic site-directed isotopically 13C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4−13C], [1,4−13C] and [2,3−13C] 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 13C 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 [1,4−13C]-succinic acid or [2,3−13C]-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 [1,4−13C]-succinic acid, while the Cα and Cβ carbons of the residues were labeled by [2,3 13C]-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 α-helical transmembrane protein structures.
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Abbreviations
- LH2:
-
light-harvesting 2
- MAS NMR:
-
magic angle spinning NMR
- PDSD:
-
proton driven spin diffusion.
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van Gammeren, A.J., Hulsbergen, F.B., Hollander, J.G. et al. Biosynthetic site-specific 13 C labeling of the light-harvesting 2 protein complex: A model for solid state NMR structure determination of transmembrane proteins. J Biomol NMR 30, 267–274 (2004). https://doi.org/10.1007/s10858-004-3736-7
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DOI: https://doi.org/10.1007/s10858-004-3736-7