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。它们也被广泛应用于生物技术行业。它们极大地提高了反应速度，具有精致的特异性，并且在水中发挥作用。利用化学工业中使用的酶的能力将是一个重大进步。不幸的是，蛋白质最重要的成分之一，它们的氢原子是看不见的。拟议工作的长期目标是解决这一问题，并使研究人员更容易获得氢原子的位置。这一结果将为利用酶工程技术设计新型催化剂提供参考。为支持采用待开发的方法，将设立一个公共网络服务器来处理用户提交的数据。此外，该项目还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。该项目还将提供纳入研究计划的新课程、外联和学生研究机会。与校园学生支持计划合作，特别强调支持夏威夷原住民和少数民族STEM学生。将开发一门以研究项目为基础的本科蛋白质生物化学课程。学生们在表达、纯化和结晶不同的铁还蛋白突变体方面的工作增加了研究努力。NG将作为少数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联活动，并培训教师带领学生探索最大限度地生长铁氧还蛋白晶体的条件。该研究使用新颖的实验和计算方法，通过将建议的计算方法Hypo(氢气预测和观测)应用于X射线和中子衍射数据，实现识别和定位蛋白质结构中的氢原子的目标。传统上，结晶学家通过与等高线电子密度图进行肉眼比较来确定原子是否存在于晶体结构中。Hypo采取了一种根本不同的量化方法，回答了这样一个问题：给出一张地图，原子的排列最有可能是什么？在合作的橡树岭国家实验室，Hypo和强大的新中子源的结合将使结构生物学家更容易获得中子结晶学。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分析将用于确定铁氧还蛋白的中子结构，以确定铁氧还蛋白是如何传递电子的。对氢原子在铁氧还蛋白中的作用的新见解将在其他电子转移蛋白质和一般蛋白质中有重要的应用。

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.