CAREER: Multidimensional High Field Solid-State NMR Methods for Protein Structure Determination

在这个CAREER项目中，PI将开发高场魔角旋转固态核磁共振（SSNMR）方法，并应用它们来理解蛋白质结构和动力学的基本方面。在过去的几年中，使用均匀- 13c，15N富集的蛋白质样品获得了小肽和蛋白质的第一个完整的SSNMR结构。该项目旨在使这些方法更加普遍适用，提高结构确定的效率和质量。本项目的智力优势集中在新颖的SSNMR方法上：(1)将建立新的化学位移分配实验和协议，采用3D和降维数据采样方案；(2)建立用于测量位分辨化学位移各向异性参数的脉冲序列，并将实验值与从头计算结果进行比较，作为主链和侧链构象的约束条件；(3)侧链构象交换将通过在一定温度范围内的化学位移和偶极光谱分析来检验；(4)在三维光谱中，通过测量侧链化学位移作为pH的函数，可以直接确定固体蛋白质中可电离残基的pKa。这些实验将提供几种目前生物物理学界无法获得的数据；例如，这些数据将有助于解释溶液核磁共振弛豫实验，并直接测试固体蛋白质的分子动力学和静电计算。此外，所开发的方法将直接适用于非可溶性蛋白，如膜蛋白和肽聚集体和原纤维。该项目的其他更广泛的影响包括为SSNMR培养新一代优秀的研究人员。研究生和本科生研究人员（来自文理学院和代表性不足的群体）将接受结构生物学和核磁共振的培训。在资助期间，每年将在主办机构举办为期三天的暑期学校。研讨会将包括研讨会、500和750兆赫光谱仪上的现代核磁共振实验演示、设施参观和核磁共振软件实践培训。每年将从学术和工业实验室中抽取12名参与者，优先考虑文理学院和小型研究型大学的教师。PI将协助这些教育工作者确定具有成本效益的策略，以升级溶液核磁共振光谱仪以进行魔角旋转实验，并以此为工作向科学界的更大截面展示现代魔角旋转ssmr。

In this CAREER project, the PI will develop high-field magic-angle spinning solid-state NMR (SSNMR) methods and apply them to understand fundamental aspects of protein structure and dynamics. In the last few years, the first complete SSNMR structures of small peptides and proteins using uniformly-13C,15N enriched protein samples have been obtained. The PI aims to make these methods more generally applicable and improve the efficiency and quality of structure determination. The intellectual merit of this project centers on novel SSNMR methodology: (1) new chemical shift assignment experiments and protocols, employing 3D and reduced dimensionality data sampling schemes, will be established; (2) pulse sequences for measuring site-resolved chemical shift anisotropy parameters will be developed, and the experimental values compared to ab initio calculations to serve as constraints of backbone and side-chain conformation; (3) side-chain conformational exchange will be examined by analysis of chemical shift and dipolar spectra over a range of temperatures; and (4) pKa's of ionizable residues in solid proteins will be directly determined by measuring side-chain chemical shifts as a function of pH in 3D spectra.These experiments will provide several types of data that are currently unavailable to the biophysics community; for example, the data will assist in the interpretation of solution NMR relaxation experiments and directly test molecular dynamics and electrostatics calculations in solid proteins. Furthermore, the methods developed will be directly applicable to non-soluble proteins, such as membrane proteins and peptide aggregates and fibrils. Additional broader impacts of this project include educating a new generation of excellent researchers for SSNMR. Both graduate students and undergraduate researchers (drawn from liberal arts colleges and under-represented groups) will be trained in structural biology and NMR. A three-day SSNMR summer school will be held at the host institution each year during the grant period. The workshop will consist of seminars, demonstrations of modern SSNMR experiments on 500 and 750 MHz spectrometers, facility tours, and hands-on NMR software training. A dozen participants each year will be drawn from academic and industrial laboratories, with priority given to faculty from liberal arts colleges and smaller research universities. The PI will assist these educators in identifying cost-effective strategies for upgrading solution NMR spectrometers to perform magic-angle spinning experiments, and as such work to expose a larger cross-section of the scientific community to modern magic-angle spinning SSNMR.