Supramolecular structural analysis of photosynthetic light-harvesting machinery

基于一种改进的重建方法，获得了细菌核心捕光复合体生物功能亚基中的细菌叶绿素(BCHL)分子的高分辨溶液核磁共振谱。重组的色素整合膜多肽亚基在室温下高浓度下稳定、均一，在818 nm处有一Q_y吸收峰。已经专门为BCHL a指定了^1H-和^&lt；13&gt；C化学位移，使用完全和选择性的^&lt；13&gt；C标记的颜料与天然丰富的多肽混合在氚洗涤剂溶液中。在重组时观察到显著的信号展宽，其中细菌素大环表现为高度受限的分子运动，而植物酚侧链保持相对移动。3^1羰基碳的^&lt；13&gt；C化学位移显示出较大的前场变化，表明所有的乙酰羰基都有很强的氢键作用。羰基…更多的碳在13^1处产生两个等强度的共振峰，这表明一个亚基中的一个Bchl a分子中的酮基形成了比另一个Bchl a分子中更强的氢键。用小角中子散射法测量了红色红螺菌LH1复合体B820亚基的内禀尺寸，并用Guinier分析进行了评估。LH1解离和重组得到的B820亚基的相对分子质量为11400±500，旋转半径为11.0±1.0Å，相当于一个由一对αβ多肽和两个细菌叶绿素a分子组成的杂二聚体。这一结果为细菌叶绿素a分子在稳定和维持B820亚基在溶液中的二聚态中起着至关重要的作用提供了证据。对单独的α-和β-多肽的进一步测量表明，这两种多肽在聚集性质上有明显的差异。-多肽似乎以单体形式均匀地溶解在OG溶液中，而β-多肽倾向于自缔合形式，即使在洗涤剂存在下也倾向于形成大的聚集体。这种聚集趋势的差异与α-和β-多肽与细菌叶绿素a分子重组的不同行为有关。我们用多维核磁共振技术测定了来自野生紫色光合细菌R.rubrum的核心捕光(LH1)α-和β-多肽的溶液结构。α-多肽的结构由中央跨膜区上的32个氨基酸组成的长螺旋和N-末端的短螺旋段组成，紧接着是一个三个残基的环。β-多肽的结构显示膜跨区有一个由32个氨基酸组成的单一螺旋。在中心螺旋区的主链酰胺质子上发现了较强的氢键，表明这两种多肽具有刚性。红色红假单胞菌LH1α-和β-多肽的总体结构与以前报道的球形红杆菌LH1β-多肽不同，但与X射线结晶学测定的相应LH2α-和β-多肽的结构非常相似。用溶液结构构建的LH1复合体结构亚单位(B820)的模型揭示了LH1α-和β-多肽之间相互作用的几个重要特征。许多实验表明，这两个多肽的N-末端区域对稳定B820和LH1复合体的意义可能归因于α-多肽的短N-末端螺旋(Trp2-Gln6)与β-多肽中的GxxxG基序之间的相互作用。较少

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