Davis C M, Bustamante P L, Todd J B, Parkes-Loach P S, McGlynn P, Olsen J D, McMaster L, Hunter C N, Loach P A
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Biochemistry. 1997 Mar 25;36(12):3671-9. doi: 10.1021/bi962386p.
Seven mutant LH1 polypeptides of Rhodobactor sphaeroides have been isolated, and their behaviors in in vitro reconstitution of LH1 and its subunit complex have been characterized. Two mutants were selected to address the increased stability of the subunit complex of Rb. sphaeroides compared with that of Rhodobacter capsulatus. We found that this difference can be largely ascribed to the existence of Tyr at position +4 in the beta-polypeptide (the numbering system used assigns position 0 to the His which provides the coordinating ligand to bacteriochlorophyll) of the former bacterium compared to Met in that position in the latter. The amount of energy involved in the increased interaction was 1.6 kcal/mol, which would be consistent with a hydrogen bond involving Tyr. Mutation of the His at position 0 to Asn allows an estimate of the binding energy for subunit formation contributed by coordination of the imidazole group of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/mol per BChl. Finally, an evaluation of the role of amino acids in the C-terminal region of the alpha-polypeptide was begun. Reconstitution of a mutant alpha-polypeptide in which Trp at position +11 was changed to Phe resulted in optimal formation of an LH1-type complex whose lambda(max) was blue-shifted to 853 nm, the same as observed in the intact bacterium harboring this mutation. These results provide further confirmation that the environment of BChl in reconstituted LH1 complexes is the same as in vivo and support the assignment of this residue to a role in hydrogen bonding with the C3(1) carbonyl group of BChl. Two other mutants of the alpha-polypeptide in which 5 and 14 amino acids in the C-terminus were deleted were also examined. These were of interest because the latter mutant, unlike the former, resulted in a low level of expression of LH1 in intact cells. However, with both of these mutant polypeptides, reconstitution appeared identical to that of the native system. In the case of the mutant shortened by 14 amino acids, a small blue-shift in lambda(max) to 861 nm was observed, again reproducing the blue-shift exhibited by the intact cells. Thus, these results suggest that the lowered levels of in vivo expression observed in these two mutants are due to reduced incorporation of the alpha-polypeptide into the membrane or its increased degradation, rather than to decreased stabilization of the LH1 complex.
已分离出球形红杆菌的七种突变型LH1多肽，并对它们在LH1及其亚基复合物的体外重组中的行为进行了表征。选择了两个突变体来研究球形红杆菌亚基复合物与荚膜红杆菌相比稳定性增加的情况。我们发现，这种差异很大程度上可归因于前一种细菌β多肽中+4位存在酪氨酸（所使用的编号系统将0位指定给为细菌叶绿素提供配位配体的组氨酸），而后一种细菌该位置为甲硫氨酸。增加的相互作用所涉及的能量为1.6千卡/摩尔，这与涉及酪氨酸的氢键相符。将0位的组氨酸突变为天冬酰胺，可以估算出组氨酸的咪唑基团与每个细菌叶绿素的细菌叶绿素镁原子配位对亚基形成的结合能>4.5千卡/摩尔。最后，开始评估α多肽C末端区域中氨基酸的作用。将+11位色氨酸变为苯丙氨酸的突变型α多肽的重组导致形成最佳的LH1型复合物，其最大吸收波长蓝移至853纳米，与携带此突变的完整细菌中观察到的相同。这些结果进一步证实了重组LH1复合物中细菌叶绿素的环境与体内相同，并支持该残基在与细菌叶绿素的C3(1)羰基形成氢键中起作用。还研究了α多肽的另外两个突变体，其中C末端分别缺失了5个和14个氨基酸。这些突变体很有趣，因为后一个突变体与前一个不同，导致完整细胞中LH1的表达水平较低。然而，对于这两种突变多肽，重组似乎与天然系统相同。对于缩短了14个氨基酸的突变体，观察到最大吸收波长有小的蓝移至861纳米，再次重现了完整细胞所表现出的蓝移。因此，这些结果表明，在这两个突变体中观察到的体内表达水平降低是由于α多肽掺入膜中的减少或其降解增加，而不是由于LH1复合物的稳定性降低。
