High Resolution Field Cycling NMR Spectroscopy as a Probe of Phospholipid Dynamics

磷脂在膜中的运动和动力学是这些渗透屏障的重要特征。由磷酸基团和甘油主链组成的界面区域可能是研究最少的，但作为与许多蛋白质和其他结合或插入膜的分子接触的极其重要的区域。利用高分辨率场循环核磁共振波谱技术测量磷脂分子中该界面区域的核磁共振弛豫速率，为表征该界面区域提供了一种新的方法。该方法是理想的生物磷酸盐和羰基在酯或酰胺键，解释松弛率往往是困难的。本研究将使用13C合成的磷脂作为一种方法，将该技术扩展到越来越复杂的混合物中。一个主要的目标将是确定胆固醇如何影响界面区域。该技术还将扩展到其他核（质子核磁共振场循环），以扩大可以分析的膜部分。用这种新的核磁共振方法研究的特定生物系统将包括小的蛋白质/磷脂圆盘，它们是脂蛋白和细胞信号传导中重要的酶PTEN的前体，PTEN的活性被认为是由磷脂结合到蛋白质上的变张位点来调节的。本研究的结果将填补我们如何描述磷脂在膜中的运动的空白，扩展这种新方法的范围，使其在其他领域更具吸引力和实用性，并提供两个重要的生物系统的具体描述，其中界面行为的详细信息不易通过其他手段获得。这个项目的广泛影响有三个组成部分。(1)培训：参与该项目的研究生和本科生（女性和少数民族继续纳入）将接受核磁共振理论和实验方面的培训。他们还将获得制备生物分子和使用模型膜的经验。这将有助于他们为从事生物技术和药理学的职业做好准备。(2)成果的传播：项目负责人和学生就项目举办的研讨会和发表的成果将展示研究方法。在这些研究中获得的运动参数将提供一个独特的数据集，用作双层计算研究的实验测试。作为一种特殊的方式来激发科学界对使用一种非常独特的资源（雷德菲尔德高分辨率野外循环器）的兴趣，PI将继续为感兴趣的科学家提供运行样本。这些相互作用将扩大该技术的使用范围，并使31P NMR重新成为探索许多系统的工具。(3)学术界以外的拓展：在更广泛的层面上，布兰迪斯大学独特的实地骑行者将成为每年波士顿学院女性科学与技术实地旅行的目的地。让高中女生（以及本科生）接触基础科学的不同方面，特别是向她们展示独一无二的设备，可以提高科学素养，但更重要的是增强她们对科学的兴趣。

The motions and dynamics of phospholipids in membranes are important characteristics of these permeability barriers. The interfacial region, composed of the phosphate group and glycerol backbone, is perhaps the least studied, but extremely important as a contact with many proteins and other molecules that bind to or are inserted into membranes. Measuring nuclear magnetic resonance (NMR) relaxation rates for nuclei in this region of the phospholipid molecule by high resolution field cycling NMR spectroscopy provides a new way to characterize this interfacial region. The method is ideal for biological phosphates and carbonyls in ester or amide linkages where interpretation of relaxation rates is often difficult. This research will use phospholipids synthesized with 13C incorporated as a way to extend the technique to increasingly complex mixtures. A major goal will be to determine how cholesterol affects the interfacial region. The technique will also be extended to other nuclei (proton NMR field cycling) to broaden the portions of a membrane that can be analyzed. The specific biological systems studied with this novel NMR methodology will include small protein/phospholipid disks that are precursors of lipoproteins and an important enzyme in cell signaling, PTEN, whose activity is thought to be regulated by phospholipids binding to an allosteric site on the protein. The results of this research will fill in gaps in how we describe motions of phospholipids in membranes, extend the scope of this new methodology to make it more attractive and useful in other fields, and provide concrete descriptions of two important biological systems where detailed information on the interfacial behavior is not easily obtained by other means. The broader impact of this project has three components. (1) Training: Both graduate and undergraduate students (with continued inclusion of women and minorities) working on this project will be trained in NMR theory and experiments. They will also obtain experience in the preparation of biomolecules and working with model membranes. This will aid in preparing them for careers in biotechnology and pharmacology. (2) Dissemination of the results: Seminars and publication of results by the PI and students on the project will showcase the methodology. Motional parameters obtained in these studies will provide a unique data set for use as an experimental test of computational studies of bilayers. As a particular way to stimulate interest in the scientific community in using a very unique resource (the Redfield high resolution field cycler), the PI will continue to offer to run samples for interested scientists. These interactions will broaden the spectrum of uses for the technique as well as revive 31P NMR as a tool for exploring many systems. (3) Outreach outside academia: On a more general level, the unique field cycler at Brandeis will be a destination for a Boston College Women in Science and Technology field trip each year. Exposing high school women (as well as undergraduates) to different aspects of basic science, particularly showing them one-of-a-kind equipment, can improve science literacy but more importantly intensify an interest in science.