Supramolecular structural analysis of photosynthetic light-harvesting machinery

基于改进的重组方法，获得了细菌核心光捕获复合物生物功能亚基中的细菌叶绿素（BChl） a分子的高分辨率溶液核磁共振光谱。在室温高浓度下，重组的色素-整体膜多肽亚基稳定且均匀，并在818 nm处表现出Q_y，吸收峰。在氘化洗涤剂溶液中，使用完全和选择性的^<13>C标记的色素与天然丰度多肽结合，专门为BChl a分配了^ 1h和^<13>C的化学位移。在重组过程中观察到显著的信号展宽，其中细菌氯大环显示在高度受限的分子运动中，而叶绿醇侧链保持相对移动。3^1羰基碳的^<13>C化学位移向下场变化较大，表明所有的乙酰羰基都有很强的氢键。更多的13^1的碳会产生两个相同强度的^<13>的C共振，这表明在一个亚基中的一个BChl a分子中的酮羰基比另一个BChl a分子中的酮羰基形成更强的氢键。用小角中子散射法测定了红红螺旋藻LH1配合物B820亚基的固有尺寸，并用Guinier分析法对其进行了评价。由LH1解离和载多肽和色素重组制备的B820亚基分子量为11400±500，旋转半径为11.0±1.0Å，对应于一对αβ-多肽和两个细菌叶绿素a分子组成的异源二聚体。结果表明，细菌叶绿素a分子在稳定和维持B820亚基在溶液中的二聚体状态中起着至关重要的作用。对α-和β-多肽的进一步测定表明，两种多肽的聚集特性有显著差异。-多肽似乎以单体形式均匀地溶解在OG溶液中，而β-多肽倾向于自结合形式，即使在洗涤剂存在的情况下也倾向于形成大的聚集体。聚集倾向的差异与α-多肽和β-多肽与细菌叶绿素a分子重构的不同行为有关。利用多维核磁共振光谱测定了野生型紫色光合细菌rrubrum核心捕光(LH1) α和β多肽的溶液结构。α-多肽的结构由中央跨膜结构域上的32个氨基酸组成的长螺旋和n端上的短螺旋段组成，其后是一个三残基环。β-多肽在跨膜区呈32个氨基酸组成的单螺旋结构。在中心螺旋区域的主酰胺质子上发现了强氢键，表明这两种多肽具有刚性性质。红草LH1 α和β-多肽的整体结构与先前报道的球形红杆菌LH1 β-多肽的结构不同，但与x射线晶体学测定的相应LH2 α和β-多肽的结构非常相似。利用溶液结构构建的LH1复合物结构亚基（B820）模型揭示了LH1 α和β-多肽相互作用的几个重要特征。许多实验表明，两种多肽的n端区域对稳定B820和LH1复合物的重要性可能归因于α-多肽的短n端螺旋（Trp2 -Gln6）与β-多肽中的GxxxG基序之间的相互作用。少

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