Collaborative Research: Design of Redox-Active Molybdenum Metalloproteins

合作研究：氧化还原活性钼金属蛋白的设计

• 批准号: 1403663
• 负责人: Brian Kuhlman
• 金额: $ 21.41万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1403663&HistoricalAwards=false
• 关键词: Collaborative; Research; Design; Redox; Active

Proposal Numbers: 1403532 / 1403663PI's: Zondlo, Neal J. / Kuhlman, BrianInstitutions: University of Delaware / University of North Carolina at Chapel HillTitle: Collaborative Research: Design of Redox-Active Molybdenum MetalloproteinsNitrogen is a critical component of DNA and proteins and is essential for all forms of life. However, most organisms cannot make use of the nitrogen gas (N2) that makes up 78% of our atmosphere. Nitrogen gas must be "fixed" to reduced forms such as ammonia (NH3) before it can be metabolized by plants and animals. This process is accomplished in nature by bacteria that live symbiotically with plants. Synthetic nitrogen fixation, performed at high temperatures and high pressures via the Haber-Bosch process, is central to the production of fertilizers, allowing the planet to sustain far larger populations than was possible prior, at the cost of 1.5% of the planet's total energy use. Nitrogenases are molybdenum-containing proteins employed by nitrogen-fixing bacteria to accomplish nitrogen fixation in water at ambient temperature and pressure, in contrast to the harsh and energy-intensive conditions of the Haber-Bosch process. Nitrogenases are also capable of reacting with carbon dioxide (i.e. conducting carbon sequestration) and carbon monoxide, reducing them to molecules that can be used as liquid fuels and feedstock chemicals. However, nitrogenases are large proteins (approximately 2000 amino acids), which prevents their application as isolated proteins. In this work, the investigators will develop synthetic proteins, only 2%-4% of the size of nitrogenase, that can bind molybdenum and react with molecules in a manner analogous to that of nitrogenases. The basis of this work is the development of novel amino acids that can both bind to molybdenum and donate electrons to the metal to engage in reaction chemistry at the molybdenum metal center, combined with computational protein design to allow the incorporation of these unnatural amino acids within a well-defined and stable protein structure that will support strong metal binding and reaction chemistry. These designed molybdenum proteins will be analyzed and characterized for their structure and ability to conduct reaction chemistry, both to understand how native molybdenum proteins can conduct important reactions and as a first step toward their potential use in processes critical to problems in energy use. This work will address critical processes central to the nitrogen cycle, the carbon cycle, energy, and sustainability, providing new insights into processes of broad fundamental importance. In the long term, this work could lead toward novel sustainable solutions to reduce energy use and synthesize feedstock chemicals from non-petroleum sources. This work will train undergraduate and graduate students in highly multidisciplinary methods, including computational protein design, peptide synthesis and characterization, organic synthesis on functional-group rich substrates, training them for integrated multidisciplinary science of the 21st century. Nitrogen fixation, the reduction of atmospheric nitrogen to ammonia, is one of the most significant processes on the planet. Nitrogen fixation is accomplished by bacteria containing the enzyme nitrogenase, a large (~2000 amino acids) enzyme containing molybdenum or vanadium and a unique iron-sulfur cluster. The accomplishment of this ancient process under mild conditions has to date never been achieved in proteins outside the native nitrogenase proteins. As inspiration, catalytic nitrogen fixation has been demonstrated using synthetic molybdenum complexes with small molecule ligands. Toward the goal of developing redox-active proteins capable of reaction chemistry similar to nitrogenase, the team will develop a synthetic molybdenum metalloprotein containing novel redox-active amino acids capable of greater electron donor ability than the native 20 amino acids. Nitrogenases and synthetic analogues accomplish dinitrogen reduction (a six-electron, six-proton process) in part due to the multiple oxidation states readily available to molybdenum (Mo(III) to Mo(VI)), plus the presence of strong electron donor ligands, including the iron-sulfur-carbide cluster. Nitrogenases and organomolybdenum complexes can also reduce electronically related compounds such as diimides (including diazine HN=NH), hydrazines (H2N-NH2), and cyanide. This work aims to develop new approaches to allow the reduction of simple compounds using synthetic proteins and provide fundamental insights into the design of catalytically active proteins. The investigators will design synthetic proteins with redox-active side chains that are not found in naturally occurring proteins and readily bind to molybdenum, vanadium and tungsten, allowing multi-electron reductions of dinitrogen and related molecules. The metal-binding properties of these designed proteins will be characterized in different metal redox states using electrochemical, biophysical, and structural methods. The designed redox-active metalloproteins will be examined for reactivity toward reduction of a series of pi-bonded compounds.This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.

建议编号：1403532 / 1403663PI：Zondlo，Neal J. / Kuhlman，BrianinStitestions：特拉华大学 /北卡罗来纳大学Chapel Hilltitle分校：协作研究：设计的设计：氧化还原活性的钼钼金属蛋白金属蛋白金属蛋白金属蛋白金属蛋白金属蛋白质的成分是DNA和蛋白质的重要​​组成蛋白，并且是全部的生命。 但是，大多数生物无法利用占我们大气中78％的氮气（N2）。氮气必须“固定”到还原形式，例如氨（NH3），然后才能被动植物代谢。这个过程是通过与植物共生的细菌在自然界中完成的。在高温下进行的合成氮固定是通过HABER-BOSCH工艺进行的，这对于肥料的生产至关重要，使地球能够维持远远超过先前的人群，而行星总能源使用的成本为1.5％。与Haber-Bosch工艺的恶劣和能量密集型条件相反，氮是氮固定细菌使用的含钼蛋白，以在环境温度和压力下在水中固定氮。氮酶还能够与二氧化碳（即导电固换）和一氧化碳反应，从而将它们降低为可以用作液体燃料和原料化学物质的分子。但是，氮酶是大蛋白（大约2000个氨基酸），可防止其用作分离的蛋白质。在这项工作中，研究者将开发合成蛋白，只有氮酶大小的2％-4％，可以结合钼并与分子反应的方式类似于氮酶。这项工作的基础是新型氨基酸的发展，这些氨基酸既可以与钼结合，又可以向金属捐赠电子在钼金属中心参与反应化学，并结合了计算蛋白设计，以允许将这些不自然的氨基酸掺入良好定义的稳定蛋白质结构中，从而支持强大的金属结合和反应化学。这些设计的钼蛋白将以它们的结构和进行反应化学的能力进行分析和表征，既了解天然钼蛋白如何进行重要的反应，又是朝着对能源使用问题至关重要的过程中潜在使用的第一步。这项工作将解决氮气周期，碳周期，能源和可持续性的关键过程，从而为广泛重要的过程提供新的见解。从长远来看，这项工作可能导致新型的可持续解决方案，以减少来自非甲基源的原料化学物质的能源利用和合成。这项工作将培训高度多学科方法的本科生和研究生，包括计算蛋白设计，肽合成和表征，对功能群富底物的有机合成，培训它们是否为21世纪的综合多学科科学培训。 氮固定，将大气中的氮还原为氨，是地球上最重要的过程之一。氮固定是通过含有酶的酶的细菌，一种含有钼或钒的大（2000氨基酸）酶以及独特的铁硫簇来完成。迄今为止，在天然氮酶蛋白之外的蛋白质中，从未实现这种古老的过程。作为灵感，使用与小分子配体的合成钼络合物证明了催化氮的固定。为了开发能够类似于硝基化化学的氧化还原活性蛋白，该团队将开发一种合成的钼金属蛋白，含有与天然20氨基酸相比，能够具有更大电子供体能力的新型氧化还原活性氨基酸。硝化酶和合成类似物可完成二氮的减少（六电子，六蛋白过程），部分原因是多种氧化态易于可用于钼（Mo（iii）到MO（VI）），加上强大的电子供体配体，包括铁 - 硫硫磺 - 硫磺 - 甲基甲壳质胶体胶合底层。氮酶和有机滋生型复合物还可以减少电子相关的化合物，例如二酰亚胺（包括二嗪HN = NH），氢氮（H2N-NH2）和氰化物。这项工作旨在开发新方法，以允许使用合成蛋白质减少简单化合物，并为催化活性蛋白的设计提供基本的见解。研究人员将设计具有自然存在的蛋白质中未发现的氧化还原活性侧链的合成蛋白质，并很容易与钼，钒和钨结合，从而允许多电子和相关分子的多电子减少。 这些设计蛋白的金属结合特性将使用电化学，生物物理和结构方法在不同的金属氧化还原态中进行表征。设计的氧化还原活性金属蛋白将被检查，以减少一系列PI键合化化合物。该奖项由CBET部门的生物技术，生物化学和生物量工程计划颁发，由分子和细胞生物学的系统和合成生物学计划共同资助。