Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 1. Minimal requirements for subunit formation
Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 1. Minimal requirements for subunit formation
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DOI：
10.1021/bi972269
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发表时间：
1998-03-10
期刊：
影响因子：
2.9
通讯作者：
Loach, PA
中科院分区：
文献类型：
--
作者：
Meadows, KA;Parkes-Loach, PS;Loach, PA
本发明描述了三种多肽中每一种的化学合成、分离和表征，所述多肽的氨基酸序列复制了类球红细菌或红色红细菌的核心捕光复合物（LH 1）的β-多肽的氨基酸序列的部分。这些生物体的LH 1天然β多肽分别含有48个和54个氨基酸。最小的合成多肽的氨基酸序列与Rb β多肽的最后16个氨基酸的氨基酸序列相同。sphaeroides（sph β 16），但在重构条件下不能形成亚基-α LH 1型复合物。此外，通过添加序列Lys-Ile-Ser-Lys在N末端上延长以增强溶解性的该多肽不能形成亚基-α LH 1型复合物。相反，含有Rb β多肽C末端的31个氨基酸的多肽。sphaeroides（sph β 31）或Rs的β-多肽的等同的31个氨基酸。红色（RR β 31）完全有能力形成亚单位型复合物，并表现出与天然β-多肽相当或超过天然β-多肽的复合物形成的缔合常数。这些亚基型复合物的吸收光谱和CD光谱与天然β-多肽形成的亚基复合物的吸收光谱和CD光谱几乎相同。可以得出结论，制备亚基复合物所需的所有结构特征都存在于定义明确的化学合成多肽中。两种多肽似乎都不与天然α-多肽相互作用形成LH 1型复合物。然而，SPH β 31形成在849 nm处吸收的LH 1型复合物，而没有α-多肽。虽然这种大小的多肽的化学合成是常见的，但跨膜区段的纯化更具挑战性，因为多肽在水中缺乏溶解性。这里报道的化学合成代表了首次这样的跨膜多肽的合成，其在重构时显示天然活性。
Described are the chemical synthesis, isolation, and characterization of each of three polypeptides whose amino acid sequences reproduce portions of the amino acid sequence of the beta-polypeptides of the core light-harvesting complex (LH1) of Rhodobacter sphaeroides or Rhodospirillum rubrum. The native beta-polypeptides of LH1 of these organisms contain 48 and 54 amino acids, respectively. The smallest synthetic polypeptide had an amino acid sequence identical to that of the last 16 amino acids of the beta-polypeptide of Rb. sphaeroides (sph beta 16) but failed to form either a subunit-alpha LH1-type complex under reconstitution conditions. Also, this polypeptide, lengthened on the N terminus by adding the sequence Lys-Ile-Ser-Lys to enhance solubility, failed to form a subunit-alpha LH1-type complex. In contrast, polypeptides containing either the 31 amino acids at the C terminus of the beta-polypeptide of Rb. sphaeroides (sph beta 31) or the equivalent 31 amino acids of the beta-polypeptide of Rs. rubrum (rr beta 31) were fully competent in forming a subunit-type complex and exhibited association constants for complex formation comparable to or exceeding those of the native beta-polypeptides. The absorption and CD spectra of these subunit-type complexes were nearly identical to those of subunit complexes formed with native beta-polypeptides. It may be concluded that all structural features required to make the subunit complex are present in the well-defined, chemically synthesized polypeptides. Neither polypeptide appeared to interact with the native alpha-polypeptides to form a LH1-type complex. However, sph beta 31 formed a LH1-type complex absorbing at 849 nm without an alpha-polypeptide. Although chemical syntheses of polypeptides of this size are common, the purification of membrane-spanning segments is much more challenging because the polypeptides lack solubility in water. The chemical syntheses reported here represent the first such syntheses of membrane-spanning polypeptides which display native activity upon reconstitution.