CAREER: Seeing the invisible: Locating hydrogen atoms in protein crystal structures

蛋白质酶是令人惊叹的催化剂，对生命系统的存在至关重要。它们也被广泛用于生物技术工业。它们能显著提高反应速率，非常特异，而且能在水中发挥作用。利用酶用于化学工业的能力将是一个重大的进步。不幸的是，蛋白质最重要的组成部分之一，它们的氢原子，是不可见的。这项工作的长期目标是解决这个问题，使研究人员更容易获得氢原子的位置。这些结果将推动通过酶工程设计新催化剂的努力。为了支持采用将开发的方法，将安装一个公共网络服务器来处理用户提交的数据。此外，该项目加强了夏威夷大学与能源部世界级设施之间的合作和培训机会。该项目还将提供新的课程，推广和学生研究的机会，被纳入研究计划。特别强调支持夏威夷土著和少数民族STEM学生与校园学生支持计划合作。将开发一个新的基于研究项目的本科蛋白质生物化学课程。学生们在表达、纯化和结晶不同铁氧还蛋白突变体方面的工作增加了研究工作。Ng将担任少数民族STEM支持计划的导师和演讲者，通过晶体生长挑战在当地高中开展外展活动，并培训教师带领学生探索最大化铁氧还蛋白晶体生长的条件。该研究使用新颖的实验和计算方法，通过应用拟议的计算方法来实现识别和定位蛋白质结构中的氢原子的目标，HyPO（氢预测和观测）到X射线和中子衍射数据。传统上，晶体学家通过与等高线电子密度图进行视觉比较来确定原子是否存在于晶体结构中。HyPO采用了一种完全不同的定量方法来回答这个问题：给定一张地图，原子的排列最有可能是什么？HyPO和橡树岭国家实验室合作的强大的新中子源的结合将使中子晶体学更容易为结构生物学家所用。HyPO将用于分析铁氧还蛋白，这是光合作用中的一种关键酶，其氢原子从未被直接观察到。HyPO分析将被应用于铁氧还蛋白的中子结构的测定，以确定铁氧还蛋白如何转移电子。对铁氧还蛋白中氢原子作用的新认识将在其他电子传递蛋白和一般蛋白质中具有重要的应用。

Protein enzymes are the amazing catalysts, essential for the existence of living systems. They are also used extensively in biotechnology industry. They dramatically increase reaction rates, are exquisitely specific and they function in water. The ability to harness enzymes for use in the chemical industry would be a major advance. Unfortunately, one of the most important components of proteins, their hydrogen atoms, are invisible. The long-term goal of the proposed work is to solve this problem and make the location of hydrogen atoms more accessible to researchers. The results will advance efforts to design new catalysts by enzyme engineering. To support the adoption of methods to be developed, a public web server to process user submitted data will be implemented. Additionally, the project strengthens collaborations and training opportunities between the University of Hawaii and world-class facilities at the Department of Energy. The project will also provide new curriculum, outreach and student research opportunities that are integrated in the research plan. Special emphasis is placed on supporting Native Hawaiian and minority STEM students in collaboration with campus student support programs. A new research project-based undergraduate protein biochemistry course will be developed. Student work on expressing, purifying, and crystallizing different ferredoxin mutants add to the research efforts. Ng will serve as a mentor and speaker to minority STEM support programs, conduct outreach activities at local high schools through the Crystal Growing Challenge and train teachers to lead students to explore conditions that maximize the growth of ferredoxin crystals.The research uses novel experimental and computational methods to attain its goal of identify and locating hydrogen atoms in protein structures by applying the proposed computational approach, HyPO (Hydrogen Prediction and Observation) to X-ray and neutron diffraction data. Traditionally, crystallographers determine whether an atom is present in a crystal structure by visual comparison with a contoured electron density map. HyPO takes a fundamentally different, quantitative approach by answering the question: What arrangement of atoms is most probable given a map? The combination of HyPO and powerful new neutron sources at collaborating Oak Ridge National Laboratory will make neutron crystallography more accessible to structural biologists. HyPO will be used to analyze ferredoxin, a key enzyme in photosynthesis whose hydrogen atoms have never been directly observed. HyPO analysis will be applied to the determination of neutron structures of ferredoxin to define how ferredoxin transfers electrons. New insight into the role of hydrogen atoms in ferredoxin will have important applications in other electron transfer proteins and to protein in general.