Sharpening up protein NMR: ultra-high resolution experiments

蛋白质是医学（如生物制药）和工业（如酶生物转化）的关键靶点和工具。核磁共振是研究溶液中这类蛋白质的重要方法，它提供了关于结构、动力学和相互作用的全面的特定位点信息——但前提是在光谱中解析了单个信号。一系列多维核磁共振技术因此被开发出来。在多个维度上扩展信号既能显著提高分辨率，又能编码结构信息；然而，nD NMR在实验时间上非常昂贵，并且产生的光谱难以可视化。现有的替代方法有时使用直接检测13C来改善信号色散，但是这些方法非常不敏感，需要13C标记和特殊的核磁共振探针。在这个项目中，我们将开发新的核磁共振实验，基于所谓的纯移位技术，既能提高分辨率，又能减少实验时间。纯位移核磁共振通过折叠多重结构，将光谱分辨率提高了2到10倍。在小分子中，这通常会在灵敏度上付出巨大的代价，但在蛋白质NMR中，我们使用选择性方法可以提高分辨率和灵敏度。新的纯移位方法对两类多肽最为重要，其中快速局部运动提供了纯移位方法所需的相对较慢的T2弛豫：小（< 8 kDa）蛋白质和肽，以及大蛋白质内部的内在无序区域。主要的实验目标是基于hsqc的实验1,2，如HNCO，其中纯转移方法特别有利于具有天然丰度13C的15n标记蛋白质。如果预期的收益得以实现，这些新的实验将允许以显着提高的分辨率记录1H-15N相关平面，并且浓度比目前直接获取13C的可能性低一个数量级以上。最初的开发工作将使用15n标记的泛素作为测试样本，然后将原型脉冲序列应用于ParG 3（一种参与质粒分离的小蛋白质，具有折叠和功能未折叠区域）和REF2-1（一种参与细胞mRNA识别的大蛋白质），以及我们实验室正在积极研究的病毒蛋白质。这项工作将在化学学院和曼彻斯特生物技术研究所之间进行，化学学院拥有12台中场超导光谱仪，曼彻斯特生物技术研究所拥有3台高场（高达800兆赫）光谱仪，配备了实际蛋白质应用所需的三共振冷冻探针。导师是核磁共振领域的世界级专家，最近的重点是纯转移方法1,2,5（主要在曼彻斯特发展），以及生物和生物技术中蛋白质的核磁共振研究。这是一个具有挑战性和重要的项目，涵盖了从物理学到生物学的所有领域。学生将接受核磁共振（GAM， MN）和溶液结构生物学（AG）的实用性培训，核磁共振（GAM）的基本理论，使用计算机控制的仪器进行新颖的实验（GAM， MN），以及先进的数据分析方法（AG， MN）。最初的三个为期8周的轮转将提供核磁共振光谱仪实际操作方面的培训，培训水平将达到开发新实验所需的水平；脉冲序列开发和数据分析所需的计算机编程技能，包括图形用户界面和宏编程；以及同位素标记蛋白质的产生。

Proteins are key targets and tools in both medicine (e.g. biopharmaceuticals) and industry (e.g. enzymic biotransformations). NMR is a vital method for the study of such proteins in solution, providing comprehensive site-specific information on structure, dynamics and interactions - but only if individual signals are resolved in the spectra. A range of multidimensional (nD) NMR techniques has therefore been developed. Spreading the signal in several dimensions both significantly increases resolution, and encodes structural information; however, nD NMR can be very costly in experiment time and the spectra produced can be difficult to visualise. Existing alternative methods sometimes use direct detection of 13C to improve signal dispersion, however these approaches are very insensitive and require both 13C labelling and special NMR probes. In this project we will develop new NMR experiments, based on so-called pure shift techniques, that are able both to improve resolution and to reduce experiment time. Pure shift NMR increases spectral resolution by a factor of 2 to 10, by collapsing multiplet structure. In small molecules this typically comes at a significant cost in sensitivity, but in protein NMR we use selective methods that can increase both resolution and sensitivity1.New pure shift methods will be of most importance to two classes of polypeptides in which fast local motion provides the relatively slow T2 relaxation needed for pure shift methods: small (< 8 kDa) proteins and peptides, and intrinsically disordered regions within larger proteins. The primary class of experiments targeted is that of HSQC-based experiments1,2 such as HNCO, where the pure shift approach is particularly favourable for 15N-labelled proteins with natural abundance 13C. If the anticipated gains are realised, these new experiments will allow 1H-15N correlation planes to be recorded at significantly enhanced resolution, and at more than an order of magnitude lower in concentration than is currently possible with direct 13C acquisition. Initial development work will use 15N-labelled ubiquitin as a test sample, with prototype pulse sequences then applied to ParG,3 a small protein involved in plasmid segregation which has both folded and functional unfolded regions, and on a larger protein REF2-1,4 implicated in recognition of cellular mRNA, as well as viral proteins which are under active investigation in our lab. The work will be divided between the School of Chemistry, which has 12 medium field superconducting spectrometers, and the Manchester Institute of Biotechnology, which houses three higher field (up to 800 MHz) spectrometers equipped with the triple-resonance cryoprobes needed for practical protein applications.The supervisors are world experts in NMR, with a recent focus on pure shift methods1,2,5 (largely developed in Manchester), and on NMR studies of proteins in biology and biotechnology. This is a challenging and important project that spans the full range from physics to biology. The student will be trained in the practicalities of NMR (GAM, MN) and of structural biology in solution (AG), the basic theory of NMR (GAM), the use of computer-controlled instrumentation to perform novel experiments (GAM, MN), and advanced data analysis methods (AG, MN). The three initial 8-week rotations will provide training in the practical operation of NMR spectrometers, at the level needed for development of new experiments; the computer programming skills needed for pulse sequence development and data analysis, including graphical user interfaces and macro programming; and the production of isotopically-labelled proteins.