Conformations of Biomolecules by Advanced Electron Paramagnetic Resonance Methods

本研究项目旨在了解多组分蛋白质系统中分子间相互作用在自组装和结构-功能关系中的基本作用。具体来说，该项目将研究特定的脂质-蛋白质相互作用如何影响跨膜蛋白质结构和动力学，以及金属离子-蛋白质相互作用如何引发病毒的大规模结构变化。该项目将使用三种病毒系统：Sindbis病毒、番茄灌木特技病毒（TBSV）和红三叶草坏死花叶病毒（RCNMV）。磷脂双层和模拟包膜病毒跨膜结构域糖蛋白的小肽之间的相互作用将进行研究，以确定对膜跨越结构域的限制，以正确整合到双层中，并阐明残基序列突变和特异性脂质-肽相互作用对肽跨膜组装和动力学的影响。本研究将阐明二价金属离子引发植物病毒大规模结构变化的分子机制和作用及其对病毒衣壳动力学的影响。这些问题将通过氮氧化物自旋标记EPR、高场EPR以及使用锰离子作为钙离子的光谱探针来解决。本项目开发的高场EPR方法将适用于其他钙结合系统的研究以及膜相关蛋白和跨膜蛋白的研究。更广泛的影响：该项目将加强NCSU研究的EPR基础设施；该项目将支持最先进的多频EPR设备的进一步发展，该设备将为化学、材料和生命科学等不同领域的研究人员提供服务。该项目将通过拟议的合作，在分子和结构生物学以及植物病理学领域开发新的高场EPR方法。该项目还将对来自代表性不足群体的学生，特别是妇女的学生进行多学科培训，以便在病毒学中应用复杂的生物物理技术。

This research project is aimed at understanding fundamental roles of intermolecular interactions in self-assembly and structure-function relationships in multicomponent protein systems. Specifically, the project will investigate how specific lipid-protein interactions can influence transmembrane protein structure and dynamics and how metal ion-protein interactions can trigger large scale structural changes in viruses. The project will use three viral systems: Sindbis virus, Tomato Bushy Stunt virus (TBSV), and Red Clover Necrotic Mosaic virus (RCNMV). Investigation of interactions between phospholipid bilayers and small peptides mimicking the transmembrane domain glycoprotein of enveloped virus will be carried out to define constraints placed upon membrane-spanning domains for correct integration into the bilayer and to elucidate the effects of residue-sequence mutations and specific lipid-peptide interactions on peptide transmembrane assembly and dynamics. The research will elucidate the molecular mechanism and the role of divalent metal ions in triggering large-scale structural changes in plant viruses and its effect on dynamics of viral capsids. Those questions will be addressed by nitroxide spin-labeling EPR, High Field EPR, and by using manganese ions as a spectroscopic probe for the calcium ions. The High Field EPR methods developed in the course of this project will be suitable for studies of other calcium-binding systems and for investigation of membrane-associated and transmembrane proteins.Broader impacts: The project will enhance the EPR infrastructure for research at NCSU; the project will support further development of the state-of-the-art multifrequency EPR facility that will be available for the researchers in diverse areas of chemistry, materials and life science. The project will develop new High Field EPR methods in areas of molecular and structural biology and plant pathology through proposed collaborations. The project will also result in multi disciplinary training of students from under represented groups, especially women, in application of sophisticated biophysical techniques in virology.