Braun P, Olsen J D, Strohmann B, Hunter C N, Scheer H
Botanical Institute, Ludwig-Maximilian University, Menzingerstrasse 67, D-80638 Munich, Germany.
J Mol Biol. 2002 May 10;318(4):1085-95. doi: 10.1016/S0022-2836(02)00192-4.
The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins.
对一种细菌光捕获复合物(来自光合细菌球形红杆菌的LH2-α)的跨膜、结合细菌叶绿素区域进行了重新设计,并在缺乏LH2的球形红杆菌突变体中进行了过表达。细菌叶绿素作为一个内部探针,用于检测这个新区域对LH2复合物组装和能量转移功能的适应性。用一个14个残基的丙氨酸-亮氨酸序列替换LH2-α的跨膜片段(缬氨酸-7到苏氨酸+6),几乎不会干扰吸收和传递光能的能力。这段序列构成了跨膜螺旋的残基,这些残基与通过α和β亚基的组氨酸配位的细菌叶绿素分子紧密接触(≤4.5 Å)。在这个丙氨酸-亮氨酸序列中,既没有替换结合细菌叶绿素的α-组氨酸0,也没有替换相邻的α-异亮氨酸-1。由具有模型跨膜序列的LH2-α和正常的LH2-β组成的新型LH2复合物在体内组装成一个复合物,其生化和光谱性质与天然复合物非常相似。相比之下,在模型跨膜螺旋C末端外额外插入四个残基会导致功能性天线复合物完全丧失。结果表明,光能可以通过仅附着在多肽上少数关键残基上的细菌叶绿素分子有效地捕获和转移,而细菌叶绿素-螺旋界面处的残基对于这种膜蛋白复合物的功能组装似乎在很大程度上是可有可无的。这种具有简化跨膜结构域以及用于组装和功能的内置探针的新型天线,为研究影响膜嵌入蛋白中生色团组装的因素提供了一个强大的模型系统。