An ODNP Probe to Study Hydration Dynamics in Membrane Protein

项目摘要/摘要 拟议的研究重点是开发用于液体状态的交钥匙仪器 Overhauser动态核极化(ODNP)光谱用于研究特定位点的翻译动力学 位于生物大分子界面的水分子，如膜蛋白。它将允许 研究人员可以轻松地在最先进的商业X波段CW/PULSE中进行ODNP实验 电子顺磁共振(EPR)光谱仪，或者在独立的台式ODNP系统中。 近年来，在核磁共振实验中，DNP已被证明是一种增强信号强度的可靠方法 在世界各地的实验室中，在使DNP用于固体和溶解方面取得了实质性进展- 状态核磁共振波谱。这一进展引起了人们对ODNP光谱学的新兴趣。尽管该方法 自20世纪60年代以来，它才被成功地应用于研究特定地点的翻译 位于膜蛋白等生物大分子界面的水的动力学。ODNP可以 绘制出膜蛋白和脂膜的局部和特定部位的水化动力学图景 并且可以提供有关蛋白质结构和动力学的关键信息。 这种方法的主要挑战之一是，目前还没有交钥匙、健壮的工具可用。 在此SBIR第2阶段应用程序中，我们将在第1阶段成功的基础上构建。我们建议发展一项持续的- 集成核磁共振仪的微波功率源WAVE ODNP谐振器及其应用 程序包为OpenVnmrJ，完全控制实验。谐振器将具有很大的转换系数 以减少所需的微波功率，从而减少样品加热。 这项技术的成功开发将为研究人员提供使用仪器的途径 允许他们将ODNP光谱学纳入他们的研究程序中，而不需要麻烦地进行故障排除 自制设备。这将极大地推广该方法，并对许多由 美国国立卫生研究院。

Project Summary / Abstract The proposed research focuses on the development of turn-key instrumentation for liquid-state Overhauser Dynamic Nuclear Polarization (ODNP) spectroscopy to study the site-specific translational dynamics of water molecules located at the interface of bio-macromolecules such as membrane proteins. It will allow researchers to readily perform ODNP experiments in a state-of-the-art commercially available X-band cw/pulsed electron paramagnetic resonance (EPR) spectrometer or, alternatively, in a standalone, benchtop ODNP system. In recent years, DNP has proven to be a robust method to increase signal intensities in NMR experiments in laboratories around the world and substantial progress has been made in adapting DNP for solid- and solution- state NMR spectroscopy. This progress has sparked a new interest in ODNP spectroscopy. Although the method is known since the 1960s it has just recently been applied successfully to study the site-specific translational dynamics of water located at the interface of large bio-macromolecules such as membrane proteins. ODNP can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes and can provide critical information about the protein structure and dynamics. One of the major challenge of this method is that currently no turn-key, robust instrumentation is available. In this SBIR phase 2 application we will build on the phase 1 success. We propose to develop a continuous- wave ODNP resonator, a microwave power source with integrated NMR spectrometer, and an application package for OpenVnmrJ to completely control the experiment. The resonator will have a large conversion factor to reduce the required microwave power and therefore sample heating. The successful development of this technology will provide researchers access to instrumentation allowing them to incorporate ODNP spectroscopy in their research routine without the hassle of troubleshooting home-built equipment. This will greatly proliferate the method and is of large interest to many projects funded by the U.S. National Institutes of Health.