High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers
High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers
复制标题
DOI：
10.1074/jbc.m111.326447
复制
发表时间：
2012-03-09
影响因子：
4.8
通讯作者：
Hanson, Deborah K.
中科院分区：
文献类型：
--
作者：
Faries, Kaitlyn M.;Kressel, Lucas L.;Hanson, Deborah K.
光合作用反应中心在一系列跨膜电子转移反应中将光能转化为化学能，每个反应的产率接近100%。反应中心的结构揭示了两个对称相关的辅因子分支(表示为A和B)，它们在功能上是不对称的；紫色细菌反应中心仅使用A途径。以前，定点突变已经产生了能够仅通过B支链辅助因子进行跨膜电荷分离的反应中心，但最佳的总电子转移产率仍然很低。为了更好地了解电子转移的方向性和产量背后的结构和能量因素，蛋白质-辅因子复合体内的位点被定向分子进化策略所针对，该策略实现了流线型突变和高通量光谱筛选。多顺反子方法能够有效地构建和表达具有高度序列相似性的两个密切调控的亚基的异寡聚复合体的大量变体，这是许多原核和真核跨膜蛋白组合的共同特征。该策略已经成功地发现了几个突变反应中心，提高了B途径的效率；它们携带着使用传统方法未被探索或连接的多个取代。这项工作扩大了我们对结构-功能关系的理解，这些关系决定了生物能量转换反应的效率，这些概念将有助于设计能够有效地分离电荷和稳定电荷的生物启发组件。
Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.