Sharpening up protein NMR: ultra-high resolution experiments

蛋白质是医学（例如生物制药）和工业（例如酶促生物转化）中的关键靶标和工具。NMR是研究溶液中这类蛋白质的重要方法，可提供有关结构、动力学和相互作用的全面位点特异性信息-但前提是光谱中的单个信号得到解析。因此，开发了一系列多维（nD）NMR技术。在多个维度上扩展信号既显着提高了分辨率，又编码了结构信息;然而，nD NMR在实验时间上可能非常昂贵，并且产生的光谱可能难以可视化。现有的替代方法有时使用13 C的直接检测来改善信号分散，然而这些方法非常不敏感，并且需要13 C标记和特殊的NMR探针。在这个项目中，我们将开发新的NMR实验，基于所谓的纯位移技术，既能够提高分辨率，又能减少实验时间。纯位移NMR通过折叠多重态结构将光谱分辨率提高2至10倍。在小分子中，这通常以灵敏度为显著代价，但在蛋白质NMR中，我们使用可以提高分辨率和灵敏度的选择性方法1。新的纯位移方法对于两类多肽最为重要，其中快速局部运动提供了纯位移方法所需的相对较慢的T2弛豫：小（< 8 kDa）蛋白质和肽，以及较大蛋白质内的固有无序区域。目标实验的主要类别是基于HSQC的实验1，2，如HNCO，其中纯位移方法特别有利于具有天然丰度13 C的15 N标记蛋白质。如果实现预期的收益，这些新的实验将允许1H-15 N相关平面被记录在显着增强的分辨率，并在一个以上的数量级低于目前可能与直接13 C采集的浓度。最初的开发工作将使用15 N标记的泛素作为测试样品，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，具有折叠和功能性未折叠区域，以及涉及细胞mRNA识别的较大蛋白REF 2 - 1，4，以及我们实验室正在积极研究的病毒蛋白。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个中场超导光谱仪，曼彻斯特生物技术研究所拥有三个更高的场（高达800 MHz）光谱仪，配备了实际蛋白质应用所需的三重共振冷冻探针。主管是NMR领域的世界专家，最近专注于纯位移方法1，2，5（主要在曼彻斯特开发），以及生物学和生物技术中蛋白质的NMR研究。这是一个具有挑战性和重要性的项目，涵盖了从物理学到生物学的全方位。学生将接受NMR（GAM，MN）和溶液结构生物学（AG）的实用性，NMR（GAM）的基本理论，使用计算机控制的仪器进行新实验（GAM，MN）和先进的数据分析方法（AG，MN）的培训。最初三次为期8周的轮调将提供以下方面的培训：核磁共振波谱仪的实际操作，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程序设计;以及同位素标记蛋白质的生产。

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.