LiT: Evolution of Asymmetry in Photosynthetic Reaction Centers

智慧价值光合成反应中心(RCS)是生命中最古老和最有用的设备之一，使生物圈能够利用持续袭击我们星球的丰富太阳能，并以不同的谋生方式多样化形成大量物种。所有已知的RCS都具有对称结构，使用两个相似(或相同)的膜插入多肽形成一个二聚体核心，该核心结合了电子通过膜转移的辅因子。这种对称的排列产生了两个辅因子分支，沿着这两个分支，光驱动的电子转移可能来自顶部的一对特殊的叶绿素(叶绿素衍生物)。每个分支的前两个成员是氯素，而第三个成员是苯二酚。众所周知，初始电子转移几乎完全发生在表征良好的2型RCS的两个分支中的一个分支上。尽管这种强烈的不对称性的起源仍然存在争议，但其中大部分可以通过这样一个事实来解释：不活跃分支的蛋白质提供的环境变得不利于电子转移。以前的努力表明，光系统I(PS1)几乎可以同等地使用它的两个分支。该项目的目的是扩大这些成果，以便了解方向性如何受到影响。核心问题是：“当电子有多种途径时，自然界如何引导电子转移？”这个问题将通过对绿藻Clamydomonas rehardtii中的核心多肽进行遗传操作来解决。一组在初级电子供体/受体对附近有突变的核心多肽将通过纳秒时间尺度上的时间分辨光学光谱进行筛选，以识别那些改变方向性的突变。随后将进行先进的生物物理分析，包括电子顺磁共振(EPR)和超快光学光谱，以确定突变对初始电荷分离事件的影响。尽管PS1是目前探索这些问题的最佳系统，但有迹象表明，它有可能创造出更好的系统。该项目包含了一个创建系统的计划，通过将同源二聚体1RC转化为异源二聚体RC来探索不对称在光合作用RCS中的应用。这将允许以受控的方式引入不对称(一次一个氨基酸残基)。计划中的方法不只是试图消除进化所造成的影响，而是探索在两条途径之间偏向电荷分离的方法，概括进化，或许探索替代机制。布罗德影响国际和平研究所的实验室有着良好的记录，将来自不同背景的本科生和研究生广泛纳入科学事业的团队成员。他们在化学、生物和物理的界面上接受跨学科科学的广泛培训。在之前的NSF奖项中，PI参与了两个新课程的开发，这两个课程将继续下去。第一个是关于生物能量机制多样性的联合讲座/实验课程，其基本目标是以与科学相同的方式教授科学，重点是探索科学文献和发展陈述技能，展示科学数据，设计和执行实验，最后撰写和评估建议。另一门课程侧重于进化论、宗教和哲学之间的关系，以研讨会形式向专业范围广泛的本科生讲授。其目的是为学生提供一个安全的空间，在其中探索关于进化论对宗教信仰和哲学/伦理立场的影响的主题。

Intellectual MeritPhotosynthetic reaction centers (RCs) are one of life's most ancient and useful devices, allowing the biosphere to exploit the abundant solar energy continuously striking our planet and to diversify into a huge number of species with distinct ways of making a living. All known RCs have symmetric structures, using two similar (or identical) membrane-inserted polypeptides to form a dimeric core, which binds the cofactors through which electrons are transferred across the membrane. This symmetric arrangement gives rise to two branches of cofactors, down which light-driven electron transfer could proceed from a 'special' pair of chlorins (chlorophyll derivatives) at the top. The first two members of each branch are chlorins, while the third is a quinone. It is known that the initial electron transfer occurs almost exclusively along one of the two branches in the well-characterized type 2 RCs. Although the origins of this strong asymmetry are still debated, much of it can be explained by the fact that the protein-provided environment of the 'inactive branch' is made less conducive for electron transfer. Previous efforts demonstrated that Photosystem I (PS1) can use both of its branches almost equally. This project is aimed at extending those results in order to understand how directionality can be influenced. The central question is: "How does nature direct electron transfer when there is more than one way for the electrons to go?" This question will be addressed by genetic manipulation of the core polypeptides in the green alga, Chlamydomonas reinhardtii. A collection of core polypeptides with mutations near the primary electron donor/acceptor pairs will be screened by time-resolved optical spectroscopy on the nanosecond timescale to identify those mutations that alter directionality. This will be followed by advanced biophysical analysis, including electron paramagnetic resonance (EPR) and ultra-fast optical spectroscopy, to determine the effect of the mutations upon the initial charge separation events. Although PS1 is currently the best system in which to explore these questions, indications are that it is possible to create a better one. The project incorporates a plan to create a system to explore the use of asymmetry in photosynthetic RCs by converting the homodimeric type 1 RC of Heliobacterium modesticaldum into a heterodimeric RC. This will allow the introduction of asymmetry in a controlled fashion (one amino acid residue at a time). Rather than merely trying to undo what evolution has wrought, the planned approaches explore ways of biasing charge separation between the two pathways, recapitulating evolution and perhaps exploring alternative mechanisms.Broader ImpactThe PI's lab has a good track record of broad inclusion of undergraduate and graduate students from diverse backgrounds as team members in the scientific enterprise. They receive a broad training in interdisciplinary science at the interface of chemistry, biology, and physics. In a previous NSF award the PI was involved in the development of two new courses, which will be continued. The first is a combined lecture/lab course on the diversity of bioenergetic mechanisms, in which the underlying goal is to teach science in the same way that science is done, with a focus on exploring the scientific literature and developing presentation skills, presenting scientific data, designing and executing experiments, and finally writing and assessing proposals. The other course focuses on the relationship between evolution, religion and philosophy, and is taught in a seminar style to undergraduates with a wide range of majors. Its purpose is to provide students a safe space in which to explore topics regarding the implications of evolutionary theory to religious beliefs and philosophical/ethical positions.