Solid-State NMR for Plant Structural Biology

Washington University, Saint Louis MO

Investigators

Abstract

Schaefer, Jacob MCB-0089905 The 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.