Investigating Membrane Proteins with Magnetic Resonance Spectroscopy

在生命过程化学项目的支持下，来自迈阿密大学的Gary A. Lorigan教授正在研究两种膜蛋白，即pinholin和gp28，它们参与细胞裂解/细胞死亡。膜蛋白约占所有已知蛋白质的三分之一，并负责生物系统的许多重要特性和功能。尽管它们丰富而重要，但关于膜相关分子的信息却很少。本项目将应用电子顺磁共振（EPR）光谱技术收集膜蛋白pinholin和gp28的结构和动态特性信息。在本项目的实施过程中，研究生和本科生将获得膜蛋白样品制备和最先进的EPR光谱技术的知识和经验。本科生将获得宝贵的生物物理化学经验，研究生将参与迈阿密大学的STEM（科学、技术、工程和数学）项目DUOS，这是他们成为优秀导师的机会。Pinholin是一种具有重要生物学意义的膜结合holin蛋白，它直接参与大肠杆菌内膜裂解导致的细胞死亡。这些研究的另一个主要目标是gp28，一种破坏噬菌体phiKT外膜（OM）的新型蛋白质。目前，关于pinholin和gp28的结构信息非常有限。嵌入在脂质双分子层中的突变pinholin的二级和三级结构将通过使用EPR光谱测量策略性放置的氮氧化物自旋标签之间的距离来阐明。最先进的EPR技术，如双电子电子共振（DEER）将被应用于研究膜结合蛋白pinholin和gp28的结构、拓扑、寡聚化状态和动态特性。我们将研究不同的构象形式。EPR数据将用于构造模型的开发，以帮助阐明井眼形成的机制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes Program, Professor Gary A. Lorigan from the Miami University is studying two membrane proteins, namely pinholin and gp28, which are involved in cell lysis/cell death. Membrane proteins make up approximately one-third of all known proteins and are responsible for many important properties and functions of biological systems. Despite their abundance and importance, there is very little information about membrane-associated molecules. This project will apply electron paramagnetic resonance (EPR) spectroscopic techniques to gather information about the structural and dynamic properties of the membrane proteins pinholin and gp28. In the implementation of this project, graduate students and undergraduate student will acquire knowledge and experience in the preparation of membrane protein samples and in state-of-the-art EPR spectroscopic techniques. Undergraduate students will obtain valuable experience in biophysical chemistry and graduate students participation in DUOS, a STEM (science, technology, engineering and mathematics) project at Miami University, is an opportunity for them to become outstanding mentors.Pinholin is a biologically significant membrane-bound holin protein that is directly involved in cell death through lysis of the inner membrane in E. coli. The other prinicipal target of these studies is gp28, a new type of protein that disrupts the outer membrane (OM) in phage phiKT. Currently, there is very limited structural information on both pinholin and gp28. The secondary and tertiary structure of mutated pinholin embedded in a lipid bilayer will be elucidated by measuring distances between strategically placed nitroxide spin labels using EPR spectroscopy. State-of-the-art EPR techniques such as double electron electron resonance (DEER) will be applied to study the structure, topology, oligomerization state, and dynamic properties of the membrane-bound proteins pinholin and gp28. Different conformational forms will be investigated. The EPR data will be used in the development of a structural model to help elucidate the mechanism of hole formation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.