CAREER: High-resolution Structural Studies of Paramagnetic Proteins by Multidimensional Solid-state NMR Spectroscopy

多维魔角旋转固态核磁共振（SSNMR）方法现在可以应用于均匀富集 13C、15N 的蛋白质，从而实现详细的原子水平研究，甚至从头测定三维分子结构。然而，该领域阻碍这些努力的突出问题之一与使用现有 SSNMR 技术可以获得的长距离（即大于 5 A）距离限制有关。在这个 CAREER 项目中，将使用固态 NMR 研究用共价结合的硝基氧自旋标签和顺磁性金属离子修饰的蛋白质，主要目标是获得传统 SSNMR 方法无法达到的长度尺度（高达约 20 A）的结构限制。具体研究目标是：（i）详细探索由偶极-偶极与不成对电子耦合引起的固相核自旋的顺磁弛豫增强（PRE），以及（ii）建立基于PRE的新型SSNMR方法，并将其应用于长距离测量、蛋白质-蛋白质相互作用的研究以及具有挑战性的生物固体中复杂NMR谱的系统简化。基于 PRE 的新型 SSNMR 方法可适用于涉及非晶体生物系统中的分子结构和相互作用的各种问题。教育推广活动的主要目标是在教育经历的早期阶段直接影响学生，特别是传统上在科学、技术、工程和数学 (STEM) 领域代表性不足的群体的学生，向他们介绍化学、生物学和物理交叉领域的当前跨学科科学，并鼓励他们以广泛的、基于发现的方式思考科学，最终目标是增加STEM 领域的学生参与度、保留率和毕业率。具体的教育推广活动包括：（i）由本科生和当地高中生组成的暑期研究实习项目；（ii）本科生实验室模块，该模块将向二年级本科生介绍现代核磁共振波谱学，并将与其他研究型实验室模块相结合；（iii）为与 PI 对蛋白质结构和功能的研究兴趣直接相关的一年级本科生的不同受众提供研讨会课程。该项目由生物科学局分子和细胞生物科学处分子生物物理学项目和数理科学局化学处实验物理化学项目共同支持。

Multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) methods can now be applied to uniformly 13C,15N enriched proteins, enabling detailed atomic-level studies and even the de novo determination of three-dimensional molecular structure. However, one of the outstanding problems in the field, which impedes these efforts, is related to the paucity of long range (i.e., greater than 5 A) distance restraints that can be obtained using the existing SSNMR techniques. In this CAREER project, proteins modified with covalently-bound nitroxide spin labels and paramagnetic metal ions, will be investigated using solid-state NMR, with the main goal of deriving structural restraints on length scales inaccessible to traditional SSNMR methods (up to ~20 A). The specific research objectives are: (i) to explore in detail the paramagnetic relaxation enhancement (PRE) of nuclear spins in the solid phase caused by the dipole-dipole coupling to unpaired electrons, and (ii) to establish novel PRE-based SSNMR methods and apply them to long-range distance measurements, studies of protein-protein interactions, and systematic simplification of complex NMR spectra in challenging biological solids. The new PRE-based SSNMR methods can be adapted to a wide variety of problems involving molecular structure and interactions in non-crystalline biological systems.The main objective of the education-outreach activities is to directly impact students in the early stage of their educational experience, particularly students from groups traditionally underrepresented in science, technology, engineering and mathematics (STEM) by introducing them to current, interdisciplinary science at the interface of chemistry, biology, and physics and encouraging them to think of science in a broad, discovery-based manner, with the ultimate goal of increasing student participation, retention and graduation rates in the STEM fields. The specific education-outreach activities include: (i) a summer research internship program with a team of undergraduate and local high-school students, (ii) an undergraduate laboratory module, which will introduce modern NMR spectroscopy to second-year undergraduates and will interface with other research-oriented laboratory modules, and (iii) a seminar course for a diverse audience of first-year undergraduate students directly related to the PI's research interests in protein structure and function. This project is jointly supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.