Investigating Membrane Proteins with Magnetic Resonance Spectroscopy

在生命过程化学项目的支持下，迈阿密大学的加里·A·洛根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，负责生物系统的许多重要性质和功能。尽管它们丰富而重要，但关于膜相关分子的信息很少。这个项目将应用电子顺磁共振(EPR)光谱技术来收集关于膜蛋白Pinholin和gp28的结构和动态性质的信息。在这个项目的实施中，研究生和本科生将获得膜蛋白样品制备和最先进的EPR光谱技术方面的知识和经验。本科生将获得宝贵的生物物理化学经验，研究生参与迈阿密大学的STEM(科学、技术、工程和数学)项目DUOS，是他们成为杰出导师的机会。Pinholin是一种具有生物学意义的膜结合Holin蛋白，通过裂解大肠杆菌内膜直接参与细胞死亡。这些研究的另一个主要目标是gp28，这是一种新型的蛋白质，可以破坏噬菌体phiKT的外膜。目前，有关Pinholin和gp28的结构信息非常有限。突变的Pinholin嵌入在脂质双层中的二级和三级结构将通过使用EPR光谱测量战略放置的氮氧化物自旋标记之间的距离来阐明。我们将利用最先进的EPR技术，如双电子电子共振(DER)，来研究膜结合蛋白Pinholin和gp28的结构、拓扑结构、齐聚状态和动力学性质。不同的构象形式将被研究。EPR数据将被用于结构模型的开发，以帮助阐明洞形成的机制。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.