Law Christopher J, Chen Jennifer, Parkes-Loach Pamela S, Loach Paul A
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Hogan Hall, Room 2-100, 2205 Tech Dr., Evanston, IL, 60208-3500, USA,
Photosynth Res. 2003;75(3):193-210. doi: 10.1023/A:1023982327748.
The protein components of the reaction center (RC) and core light-harvesting (LH 1) complexes of photosynthetic bacteria have evolved to specifically, but non-covalently, bind bacteriochlorophyll (Bchl). The contribution to binding of specific structural elements in the protein and Bchl may be determined for the LH 1 complex because its subunit can be studied by reconstitution under equilibrium conditions. Important to the determination and utilization of such information is the characterization of the interacting molecular species. To aid in this characterization, a fluorescent probe molecule has been covalently attached to each of the LH 1 polypeptides. The fluorescent probes were selected for optimal absorption and emission properties in order to facilitate their unique excitation and to enable the detection of energy transfer to Bchl. Oregon Green 488 carboxylic acid and 7-diethylaminocoumarin-3-carboxylic acid seemed to fulfill these requirements. Each of these probes were utilized to derivatize the LH1 beta-polypeptide of Rhodobacter sphaeroides. It was demonstrated that the beta-polypeptides did not interact with each other in the absence of Bchl. When Bchl was present, the probe-labeled beta-polypeptides interacted with Bchl to form subunit-type complexes much as those formed with the native polypeptides. Energy transfer from the probe to Bchl occurred with a high efficiency. The alpha-polypeptide from LH 1 of Rb. sphaeroides and that from Rhodospirillum rubrum were also derivatized in the same manner. Since these polypeptides do not oligomerize in the absence of a beta-polypeptide, reversible binding of a single Bchl to a single polypeptide could be measured. Dissociation constants for complex formation were estimated. The relevance of these data to earlier studies of equilibria involving subunit complexes is discussed. Also involved in the photoreceptor complex of Rb. sphaeroides and Rhodobacter capsulatus is another protein referred to as PufX. Two large segments of this protein were chemically synthesized, one reproducing the amino acid sequence of the core segment predicted for Rb. sphaeroides PufX and the other reproducing the amino acid sequence predicted for the core segment of Rb. capsulatus PufX. Each polypeptide was covalently labeled with a fluorescent probe and tested for energy transfer to Bchl. Each was found to bind Bchl with an affinity similar to the affinity of the LH 1 polypeptides for Bchl. It is suggested that PufX binds Bchl and interacts with a Bchlcalpha-polypeptide component of LH 1 to truncate, or interupt, the LH 1 ring adjacent to the location of the Q(B) binding site of the RC.
光合细菌反应中心（RC）和核心光捕获（LH 1）复合体的蛋白质组分已经进化到能够特异性但非共价地结合细菌叶绿素（Bchl）。对于LH 1复合体，可以确定蛋白质和Bchl中特定结构元件对结合的贡献，因为其亚基可以在平衡条件下通过重组进行研究。确定和利用此类信息的重要前提是对相互作用的分子种类进行表征。为了辅助这种表征，已将荧光探针分子共价连接到每个LH 1多肽上。选择荧光探针以获得最佳的吸收和发射特性，以便促进其独特的激发，并能够检测到向Bchl的能量转移。俄勒冈绿488羧酸和7-二乙氨基香豆素-3-羧酸似乎满足这些要求。这些探针中的每一种都用于衍生化球形红杆菌的LH1β-多肽。结果表明，在没有Bchl的情况下，β-多肽彼此不相互作用。当存在Bchl时，探针标记的β-多肽与Bchl相互作用形成亚基型复合体，与天然多肽形成的复合体非常相似。从探针到Bchl的能量转移效率很高。球形红杆菌LH 1的α-多肽和红螺菌的α-多肽也以相同的方式进行衍生化。由于这些多肽在没有β-多肽的情况下不会寡聚，因此可以测量单个Bchl与单个多肽的可逆结合。估计了复合体形成的解离常数。讨论了这些数据与早期涉及亚基复合体平衡研究的相关性。球形红杆菌和荚膜红细菌的光感受器复合体中还涉及另一种称为PufX的蛋白质。该蛋白质的两个大片段通过化学合成得到，一个再现了预测的球形红杆菌PufX核心片段的氨基酸序列，另一个再现了预测的荚膜红细菌PufX核心片段的氨基酸序列。每个多肽都用荧光探针进行共价标记，并测试其向Bchl的能量转移。发现它们与Bchl的结合亲和力与LH 1多肽对Bchl的亲和力相似。有人提出，PufX结合Bchl并与LH 1的Bchlα-多肽组分相互作用，以截断或中断与RC的Q(B)结合位点位置相邻的LH 1环。
