Conformations of Biomolecules by Advanced Electron Paramagnetic Resonance Methods

知识价值。该项目旨在通过开发新的实验能力来研究分子间相互作用在多组分生物系统中自组装和结构-功能关系中的基本作用，从而扩大电子顺磁共振（EPR），特别是高分辨率高场（HF） EPR方法的应用范围。重点将放在阐明脂质-蛋白相互作用的Sec14蛋白家族。在这个由nsf资助的项目的前期，开发了一种基于高频epr的方法来分离自旋标记蛋白残基上溶剂效应的两个主要组分，并检测氢键形成和局部静电效应。虽然膜蛋白折叠和热力学稳定性的一般概念开始出现，但用于评估局部蛋白质静电和局部氢键相互作用的实验光谱方法仍然严重有限。本研究项目旨在进一步发展自旋标记HF EPR和双共振方法，在不需要制备高质量晶体的情况下绘制蛋白质系统的氢键环境。该方法依赖于将基于氮氧化物自由基的小分子标签结合到蛋白质结构中。这些标记具有与天然蛋白质侧链相似的分子体积和结构，因此，已知只会对三级结构造成最小的扰动。这种标签的主要优点在于其EPR光谱对局部静电环境和氢键形成的敏感性，从而获得难以获得的重要生物物理数据。正在开发的高场和双共振EPR方法进一步提高了氮氧化物自旋标记对局部静电和氢键的灵敏度。在本项目中，这些方法将进一步完善，以阐明Sec14蛋白及其类似物调节磷脂代谢和膜运输之间界面的有趣且大部分未知的分子机制。自旋标记EPR和一系列互补的生物物理方法将用于研究Sec14如何识别、结合和运输磷脂，以及它如何与磷脂膜相互作用。更广泛的影响。本项目中开发的方法将填补EPR现有实验能力的空白，并使局部静电和氢键的详细生物物理研究成为可能，这些研究直接涉及许多当代感兴趣的生物系统的结构-功能关系-从模型膜和肽到离子通道，转运体和g蛋白偶联受体。该项目将整合研究和教学，在PI开发的以讲授为主的课程“生物化学中的物理方法”中加入研究驱动的实验任务。在这个项目的过程中，研究生和本科生将接受包括生物物理光谱学、化学合成和分子生物学方法在内的多个学科的培训。该项目将通过研究生教育和教授联盟（AGEP）和本科生研究经验（REU）项目扩大本科生，特别是少数民族学生的研究机会。为了向北卡罗莱纳州农村地区的本科院校伸出援助之手，PI将继续与北卡罗莱纳州彭布罗克大学合作。PI和她的团队将参与与NCSU相关的百年中学的科学项目和示范，这些项目将通过北卡罗来纳州K-12外展计划机构科学之家进行协调。

Intellectual merit. This project is aimed at expanding the arsenal of Electron Paramagnetic Resonance (EPR) and, especially, high resolution high field (HF) EPR methods by developing novel experimental capabilities to study fundamental roles of intermolecular interactions in self-assembly and structure-function relationships in multi component biological systems. Specific emphasis will be put on elucidating lipid-protein interactions for the Sec14 protein family. During the preceding phase of this NSF-funded project, an HF EPR-based method to separate two major components of solvent effects on spin-labeled protein residues and to detect hydrogen bond formation and local electrostatic effects was developed. While the general concepts of membrane protein folding and thermodynamic stability are beginning to emerge, the arsenal of experimental spectroscopic methods for assessing local protein electrostatics and local hydrogen bonding interactions remains severely limited. This research project aims at further developing spin-labeling HF EPR and double-resonance methods for mapping the hydrogen bonding environment for protein systems without the necessity of preparing high quality crystals. The method relies on incorporating small molecular tags, based on nitroxide radicals, into the protein structure. These labels have molecular volume and structure similar to the native protein side chain and, therefore, are known to cause only minimal perturbation to the tertiary structure. The main advantage of such labels lies in the sensitivity of their EPR spectra to the local electrostatic environment and hydrogen bond formation resulting in essential biophysical data that are difficult to obtain otherwise. The sensitivity of nitroxide spin labels to local electrostatics and hydrogen bonding is further enhanced by high field and double-resonance EPR methods under development. These methods will be further refined during this project to elucidate an intriguing and largely unknown molecular mechanism by which the Sec14 protein and its analogs regulate the interface between phospholipid metabolism and membrane trafficking. Spin label EPR and a complementary array of biophysical methods will be used to study how Sec14 recognizes, binds and transports phopholipids and how it interacts with phospholipid membranes.Broader impact. The methods developed in the course of this project will fill the gap in the existing experimental capabilities of EPR and enable detailed biophysical studies of local electrostatics and hydrogen bonding that are directly involved in structure-function relationships of many biological systems of contemporary interest - from model membranes and peptides to ion channels, transporters and G-protein coupled receptors. The project will integrate research and teaching by adding research-driven experimental tasks to a largely lecture course "Physical Methods in Biological Chemistry" developed by the PI. In the course of this project graduate and undergraduate students will be trained across several disciplines including biophysical spectroscopy, chemical synthesis and methods of molecular biology. The project will expand research opportunities for undergraduate students, especially, minority students through the Alliances for Graduate Education and the Professoriate (AGEP) and Research Experience for Undergraduates (REU) programs. In an effort to reach out to undergraduate colleges in rural North Carolina, the PI will continue collaboration with the University of North Carolina-Pembroke. The PI and her group will be engaged in science projects and demonstrations in the Centennial Middle School associated with NCSU that will be coordinated through the Science House, a North Carolina institution for K-12 outreach program.