Supramolecular structural analysis of photosynthetic light-harvesting machinery

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High-resolution solution NMR spectra have been obtained for the bacteriochlorophyll(BChl) a molecules in a biologically functional subunit of bacterial core light-harvesting complex based on a modified reconstitution method. The reconstituted subunit of pigment-integral membrane polypeptides is stable and homogeneous at high concentrations at room temperature, and exhibits a Q_y, absorption peak at 818 nm. ^1H-and ^<13>C chemical shifts have been specifically assigned for the BChl a using the fully and selectively ^<13>C-labeled pigments incorporated with natural abundance polypeptides in deuterated detergent solution. Remarkable signal broadening has been observed upon the reconstitution, where the bacteriochlorin macrocycle is shown in a highly restricted molecular motion while the phytol side chain remains relatively mobile. The ^<13>C chemical shift of 3^1 carbonyl carbon shows a large change to downfield, indicating a strong hydrogen bonding for all the acetyl carbonyls. Carbonyl … More carbons at 13^1 give rise to two ^<13>C resonances with equal intensities, suggesting that the keto carbonyl in one BChl a molecule within a subunit forms a stronger hydrogen bond than that in another BChl a molecule.Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11400±500 and radius of gyration of 11.0±1.0Å, corresponding to a heterodimer consisting of one pair αβ-polypeptides and two bacteriochlorophyll a molecules. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual α-and β-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The -polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the β-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was related to the different behavior between α-and β-polypeptides in reconstitution with bacteriochlorophyll a molecules.We have determined the solution structures of the core light-harvesting (LH1) α-and β-polypeptides from wild-type purple photosynthetic bacterium R.rubrum using multidimensional NMR spectroscopy. The structure of α-polypeptide consists of a long helix of 32 amino acids over the central transmembrane domain and a short helical segment at the N-terminus that is followed by a three-residue loop. The structure of β-polypeptide shows a single helix of 32 amino acids in the membrane-spanning region. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R.rubrum LH1 α-and β-polypeptides are different from those previously reported for the LH1 β-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 α-and β-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 α-and β-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2 -Gln6) of α-polypeptide and a GxxxG motif in the β-polypeptide. Less