Solid-State NMR for Plant Structural Biology

Schaefer，JacobMCB-0089905本研究的主要目标是将固体核磁共振应用于植物生物物理学中的三个重要问题：(1)鉴定二氧化碳固定酶、核酮糖-1，5-二磷酸羧基加氧酶或Rubisco的可能的变构结合部位；(2)表征具有启动子DNA结合区的调节激素-蛋白质复合体的局部构象；以及(3)确定植物细胞壁蛋白和果胶的交联机理。用于解决所有这三个问题的主要技术将是旋转回波双共振(REDOR)检测的稳定同位素标记。REDOR方法已经开发了十年。这一项目代表了一种努力，以扩大REDOR的适用范围，以解决比以前尝试过的更困难的问题。该实验室目前拥有四台适用于红色或结构生物学问题的多频固态核磁共振光谱仪，另有两台正在等待建造资金的提案。大多数植物科学都是在工业或学术生物实验室完成的，那里无法获得固态核磁共振。这项工作的第二个目标是让植物科学界意识到固态核磁共振解决结构生物学问题的能力。这项研究的长期社会意义是安全、高效地生产更多的食物。植物为人类提供了大约90%的卡路里和80%的蛋白质。至少在过去的13,000年里，这一点一直是正确的，未来也很可能继续如此。现代农业中生物技术的一个经常被提及的目标是提高作物产量，以满足饥饿人口的需求。在许多情况下，饥饿的人生活在首次开垦的边际可耕地上。在这种情况下，最重要的是Rubisco对碳的同化效率。Rubisco催化底物糖和CO2转化为-CH2O-(光合作用和碳同化)，以及底物糖和O2转化为CO2(光呼吸)。这种破坏性的竞争是不可避免的，因为Rubisco的主要反应位点的性质，它在所有光合作用的细菌、藻类和植物中都高度保守。然而，经过数十亿年的进化，高等植物已经能够区分光呼吸途径而支持碳同化途径，尽管O2已经增长到空气的21%，而CO2只有0.03%。因此，植物需氧生长，而光合细菌只能厌氧生长。植物对Rubisco的控制可能包括由一个或多个次要位点上的非底物小分子结合引起的初级位点活性的变化。第一个研究目标是通过使用15N{31P}红色的均匀标记的15N在不同的糖磷酸盐存在下的体外和体内实验来证明这种变构效应的存在。光合作用/光呼吸的变构控制的存在可能为改变大豆等植物的光呼吸控制提供了可能性，即通过Rubisco次生位点的遗传修饰来改变光呼吸控制。这种努力的动机将是使植物适应预计在未来常见的高温、低水和高外部二氧化碳的条件。其目的是证明固体核磁共振可以揭示晶体结构中看不到的变构结合部位的结构细节，因此固体核磁共振具有指导生物技术改变植物光合作用/光呼吸选择性以改善碳同化的潜力。美国数百万英亩的农田目前种植着转基因大豆、玉米、土豆和棉花，现在支持持续表达外来蛋白质产品(除草剂分解代谢酶和杀虫剂)的植物。通过应用无害的可扩散信号分子来开启/关闭表达的转基因植物是可能的。这项研究的第二个目标是开发固态核磁共振技术，用于指导调控开关的设计。最后，我们认识到，有用的转基因作物必须是一个高度集成的碳、氮和水管理系统，具有机械健全的结构。这项研究的第三个目标是使用固态核磁共振来了解植物细胞壁的结构。

Schaefer, JacobMCB-0089905The primary goal of this research is the application of solid-state NMR to three problems of importance in plant biophysics: (1) the identification of possible allosteric binding sites of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase or Rubisco; (2) the characterization of the local conformation of a regulatory hormone-protein complex with a promoter DNA binding region; and (3) the determination of mechanisms of cross-linking for plant cell-wall proteins and pectins. The principal technique to be used to solve all three problems will be stable-isotope labeling with rotational-echo double-resonance (REDOR) detection. REDOR methods have been under development now for a decade. This project represents an effort to extend the range of applicability of REDOR to more difficult problems than have yet been attempted. This laboratory presently has four multi-frequency solid-state NMR spectrometers suitable for REDOR structural biology problems, and a proposal pending for funding to build two more. Most plant science is done in industrial or academic biology laboratories where there is no access to solid-state NMR. A secondary goal of this work is to make the plant-science community aware of the capabilities of solid-state NMR to solve problems in structural biology.The long-range societal importance of this research is the safe, efficient production of more food. Plants provide approximately 90% of the calories and 80% of the protein for human consumption. This has been true for at least the last 13,000 years and is likely to continue to be true in the future. An often-stated goal of biotechnology in modern agriculture is the improvement in crop yield to feed the hungry. In many cases the hungry live on marginally arable land coming under cultivation for the first time. Of prime importance in this situation is the efficiency of the carbon assimilation by Rubisco. Rubisco catalyzes the conversion of substrate sugar and CO2 to -CH2O- (photosynthesis and carbon assimilation), and of substrate sugar and O2 to CO2 (photorespiration). This destructive competition is inevitable because of the nature of the primary reaction site of Rubisco, which is highly conserved in all photosynthetic bacteria, algae, and plants. After several billion years of evolution, however, higher plants have become capable of discriminating against the photorespiratory pathway in favor of the carbon assimilation pathway, even though O2 has grown to 21% of air and CO2 is only 0.03%. Thus, plants grow aerobically whereas photosynthetic bacteria only grow anaerobically. The control of Rubisco by plants may include alterations in the activity of the primary site caused by non-substrate small-molecule binding at one or more secondary sites. The first research goal is to demonstrate the presence of such an allosteric effect by in vitro and in vivo experiments using 15N{31P} REDOR of uniformly 15N-labeled Rubisco in the presence of various sugar phosphates. The existence of allosteric control of photosynthesis/photorespiration might offer the possibility of altering photorespiratory control in plants like soybeans by genetic modification of secondary sites of Rubisco. The motivation for such an effort would be to adapt the plant to the conditions of high temperature, low water, and high external CO2 expected to be common in the future. The goal is to demonstrate that solid-state NMR can reveal structural details of allosteric binding sites not seen in crystal structures, and that solid-state NMR therefore has the potential to direct biotechnology in the modification of the photosynthesis/photorespiration selectivity of plants for improved carbon assimilation.The millions of acres of farmland in the US currently planted with transgenic soybeans, corn, potatoes, and cotton now support plants that express foreign protein products (herbicide catabolizing enzymes and insecticides) continually. Transgenic plants with expression switched on/off by application of an innocuous diffusable signal molecule are possible. The second goal of this research is to develop solid-state NMR technology that can be used to help direct the design of the regulatory switch.Finally, we recognize that a useful transgenic crop plant must be a highly integrated system of carbon, nitrogen, and water management in a mechanically sound structure. The third goal of this research is to use solid-state NMR to gain an understanding of the architecture of the plant cell wall.