LiT: Evolution of Asymmetry in Photosynthetic Reaction Centers

光合作用反应中心（RCs）是生命中最古老和最有用的设备之一，使生物圈能够利用不断撞击我们星球的丰富太阳能，并以不同的方式多样化为大量的物种谋生。所有已知的RC都具有对称结构，使用两个相似（或相同）的膜插入多肽形成二聚体核心，其结合辅助因子，电子通过辅助因子跨膜转移。这种对称的排列产生了两个辅因子分支，光驱动的电子转移可以从顶部的一对“特殊”的二氢卟酚（叶绿素衍生物）进行。每个分支的前两个成员是二氢卟酚，而第三个是醌。它是已知的，初始电子转移发生几乎完全沿着的两个分支之一，在良好的特征2型RC。虽然这种强烈的不对称性的起源仍然存在争议，但其中大部分可以解释为这样一个事实，即蛋白质提供的“非活性分支”环境不利于电子转移。先前的研究表明，光系统I（PS1）几乎可以同等地使用它的两个分支。该项目旨在扩展这些结果，以了解方向性如何受到影响。核心问题是：“当电子有不止一条路可走时，自然界如何引导电子转移？“这个问题将通过对绿色衣藻（Chlamydiumreinhardtii）核心多肽的遗传操作来解决。将通过纳秒时间尺度上的时间分辨光谱法筛选在初级电子供体/受体对附近具有突变的核心多肽的集合，以鉴定改变方向性的那些突变。随后将进行先进的生物物理分析，包括电子顺磁共振（EPR）和超快光谱，以确定突变对初始电荷分离事件的影响。虽然PS1是目前探索这些问题的最佳系统，但有迹象表明，有可能创造一个更好的系统。该项目纳入了一个计划，以创建一个系统，探索使用不对称的光合RC转换成异二聚体RC的Heliobacterium modesticaldum的同型二聚体1型RC。这将允许以受控的方式引入不对称性（一次一个氨基酸残基）。计划中的方法不仅试图消除进化造成的影响，还探索了在两种途径之间偏置电荷分离的方法，概括了进化，也许还探索了替代机制。更广泛的影响PI的实验室有着良好的记录，广泛吸收了来自不同背景的本科生和研究生作为科学企业的团队成员。他们在化学，生物学和物理学的界面上接受跨学科科学的广泛培训。在以前的NSF奖PI参与了两个新的课程，这将继续发展。第一个是关于生物能量机制多样性的综合讲座/实验室课程，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演讲技巧，展示科学数据，设计和执行实验，最后撰写和评估提案。另一门课程侧重于进化论，宗教和哲学之间的关系，并以研讨会的形式教授给各种专业的本科生。它的目的是为学生提供一个安全的空间，在其中探索有关进化理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.