Resolution and Reconstitution of Photosystem I in Cyanobacteria and Higher Plants; Molecular Biological and Physiochemical Studies

本研究旨在研究蓝藻和绿色植物中光系统I反应中心复合体的结构、功能和组织。我们将P700-FA/FB复合体分解为P700-FX核心、P700-A1核心和P700-A0核心的能力，以及我们用改变的辅因子和转基因多肽重组每个制剂的能力，在光系统I方面提供了新的研究机会。该计划基于多肽和辅因子分解和重建光系统I反应中心的能力，并有五个主要目标：1)确定FA和/或FB参与NADP+的光还原。2)确定PsaA和PsaB上Fx铁硫簇的配体。3)确定PSAD、PSAE和PSAF在光系统I功能中的作用。4)求解PSAC、PSAD和PSAE蛋白的三维结构。5)测定重组对苯二酚的光谱性质。绿色植物能够利用阳光作为能源，利用光合作用反应中心为有机体的生长、新陈代谢和繁殖提供动力。绿色植物的叶绿体中有两种不同的反应中心，每个反应中心都含有将太阳能转化为化学能所必需的叶绿素、蛋白质和金属离子。在过去的几年里，我们已经学会了如何从其中一个反应中心去除蛋白质和金属，我们已经学会了如何从组成部分重建反应中心。在这项提案中，我们将使用现代基因工程技术改变的蛋白质来重建反应中心。我们将通过与几位分子遗传学家的密切合作来获得这些蛋白质。然后，我们将使用复杂的测量技术来确定单个蛋白质在光化学能量转换过程中的功能。通过改变一种蛋白质，同时保留其他蛋白质的功能，我们将能够确定哪种蛋白质在光合作用过程中提供一定的功能。因此，这项提议依赖于两种非常强大但截然不同的技术--遗传学和生物物理学--的结合，以解决绿色植物光合作用这一复杂问题。

This research program aims at studying the structure, function and organization of the Photosystem I reaction center complex in cyanobacteria and green plants. Our ability to resolve P700- FA/FB complex into the P700-FX core, the P700-A1 core, and P700-A0 core, and our ability to reconstitute each preparation with altered cofactors and genetically-modified polypeptides, has provided new research opportunities in Photosystem I. The proposed program is based on the ability to resolve and reconstitute the Photosystem I reaction center in terms of polypeptides and cofactors, and has five major objectives: 1) To determine the involvement of FA and/or FB in the photoreduction of NADP+. 2) To confirm the ligands to the FX iron-sulfur cluster on PsaA and PsaB. 3) To determine the roles of PsaD, PsaE and PsaF in Photosystem I function. 4) To solve the 3-dimensional structure of the PsaC, PsaD and PsaE proteins. 5) To measure the spectroscopic properties of A1 with reconstituted quinones. %%% Green plants are able to utilize sunlight as an energy source with the use of photosynthetic reaction centers to power the organism's growth, metabolism, and reproduction. Two different kinds of reaction centers are located in the chloroplasts of green plants, and each contains chlorophyll, proteins, and metals ions necessary for transforming solar energy into chemical energy. In the last several years, we have learned how to remove the proteins and metals from one of these reaction centers, and we have learned how to rebuild the reaction centers from the constituent parts. In this proposal, we will rebuild the reaction centers using proteins that have been altered using techniques of modern genetic engineering. We will obtain these proteins through a close collaboration with several molecular geneticists. We will then use sophisticated measuring techniques to determine the function of the individual proteins in the process of photochemical energy conversion. By altering one protein while retaining the function of the others, we will be able to determine which protein provides a certain function in the process of the photosynthesis. The proposal, therefore, relies on the marriage of two very powerful, but disparate techniques, genetics and biophysics, to solve the complicated problem of green plant photosynthesis.