Structure Determination of Membrane Proteins in Aligned Discoidal Lipid Bilayers by Solid-State NMR

膜蛋白在活细胞中具有多种重要的生物学功能，包括信号转导、能量转换、分子识别和离子转运。 然而，尽管膜蛋白在生物学中具有首要的重要性，但其结构的数量仍然相对稀少。 还应该强调的是，这些结构中的许多结构是在与活细胞中遇到的条件非常不同的条件下确定的（如低温、几乎不存在脂质分子、高蛋白质堆积密度）。相比之下，核磁共振（NMR）是一种微创但信息量非常丰富的技术，可以在接近生理条件下研究膜蛋白。目前的研究将集中在开发天然的膜模拟物和新的光谱方法，这将允许一个确定膜蛋白的结构下充分水合，高脂蛋白比，和生理温度下的固态NMR。作为该项目的更广泛的影响，跨学科的研究项目将提供给不同的学生群体，这项研究的结果将被推广到研究生课程和科学，技术，作为北卡罗来纳州州立大学凯南研究员计划的一部分，通过高中教师的暑期导师进行工程和数学（STEM）教育。该项目将开发基于聚合物的盘状脂质模拟物，稳定的脂质纳米盘将从根本上提高光谱分辨率，由于其特殊的磁性对齐和快速旋转扩散围绕其轴。 纳米盘的磁性排列将通过微调其直径和添加镧系元素螯合物质来增强其磁性来实现。盘状膜模拟物将被应用于包括蛋白视紫红质和人乙酰胆碱受体在内的多位膜蛋白的结构测定，这些蛋白在传统的排列脂质双层如磁性排列的双胞中表现出不足的分辨率。 将开发新的脉冲序列用于选择性激发，以减少NMR相关谱中的峰值拥挤。此外，将开发多维光谱分析的计算方法，包括自动光谱分配算法，以简化结构测定过程。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Membrane proteins perform a variety of important biological functions including signal transduction, energy conversion, molecular recognition, and ion transport in the living cells. Despite the primary importance of membrane proteins in biology, however, the number of their solved structures remains relatively scarce. It should be also emphasized that many of these structures have been determined under the conditions that were very distinct from those encountered in the living cells (such as cryogenic temperatures, near absent lipid molecules, high protein packing density). By contrast, Nuclear Magnetic Resonance (NMR) is a minimally invasive yet an exceptionally informative technique which makes it possible to study membrane proteins under nearly physiological conditions. The present research will focus on the development of native-like membrane mimetics and new spectroscopic methods, which will allow one to determine structures of membrane proteins under full hydration, high lipid-to-protein ratios, and physiological temperature by solid-state NMR. As broader impact of this project, interdisciplinary research projects will be offered to a diverse group of students and the results of this research will be promoted to graduate curricula and Science, Technology, Engineering and Mathematics (STEM) education via summer mentorship of high school teachers as part of the Kenan Fellows Program at North Carolina State University.The project will develop discoidal lipid mimetics based on polymer-stabilized lipid nanodiscs that will radically improve spectral resolution due to their exceptional magnetic alignment and fast rotational diffusion about their axes. The magnetic alignment of the nanodiscs will be achieved by fine-tuning their diameter and adding lanthanide-chelating species to enhance their magnetic properties. The discoidal membrane mimetics will be applied to structure determination of polytopic membrane proteins including proteorhodopsin and human acetylcholine receptor, which exhibit insufficient resolution in the traditional aligned lipid bilayers such as magnetically aligned bicelles. New pulse sequences will be developed for selective excitation to decrease peak crowdedness in the NMR correlation spectra. Moreover, computational methods for the analysis of multidimensional spectra will be developed including automated spectroscopic assignment algorithms to streamline the structure determination process.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.