Characterising structure, interactions and dynamics of large molecular machines and intrinsically disordered proteins using novel carbon-detected NMR

在核磁共振（NMR）光谱学中，使用专门设计的无线电波（脉冲序列）激发质子、碳和氮等核磁化，由此产生的无线电波从样品中传输，为研究人员提供有关所讨论的核的局部环境及其运动特性的信息。因此，核磁共振光谱通常是阐明分子结构、灵活性和相互作用的首选工具，因为核磁共振在原子分辨率上提供了分子动力学和相互作用的描述。本研究的主要目的是探索和发展生物分子核磁共振波谱的相关途径，即碳检测核磁共振波谱。我们的新应用程序将使我们能够可视化大分子机器的运动和机制。在蛋白质复合物、大分子机器和内在无序蛋白质的研究领域中，核磁共振波谱的主要应用依赖于质子磁化的检测。这是因为质子的磁场强度比碳强4倍，比氮强10倍，因此产生更强的核磁共振信号，更容易被探测到。然而，控制核磁共振信号线宽的自旋物理和核相互作用也依赖于相互作用核的磁场强度，因此研究人员一直在分子机器中稀释质子，以锐化核磁共振信号，避免信号重叠，从而促进新的特征。该提案的其他人和申请人最近显示了使用碳检测核磁共振光谱研究大蛋白质和内在无序蛋白质的潜力。虽然碳探测到的核磁共振信号比质子探测到的信号弱，但我们计划利用我们正在申请的新设备进一步开发和发展许多好处。首先，碳信号通常比相应的质子信号更清晰——因为它们的磁场强度较低。其次，碳信号之间的平均距离远高于质子信号之间的平均距离，从而导致信号的重叠明显减少，便于对底层参数的分析。总的来说，这意味着我们将能够监测蛋白质在其结构（蛋白质折叠）形成过程中的运动和相互作用，大分子机器，如细胞蛋白质生产机器（核糖体）和DNA重塑酶（组蛋白去乙酰化酶）。我们在应用和开发核磁共振光谱的新方法方面都有出色的记录，也在碳检测核磁共振领域。这项研究将在伦敦大学学院（UCL）结构与分子生物学研究所（ISMB）进行。一个最先进的和刺激的研究环境，专门的核磁共振机器，优化碳核的检测，高度协作的环境和世界一流的专业知识开辟了许多富有成效的跨学科合作和许多新的应用的可能性。

In nuclear magnetic resonance (NMR) spectroscopy nuclear magnetisations, such as proton, carbon and nitrogen, are excited using specifically designed radio waves (pulse sequences) and the resulting radio wave transmitted from the sample provides the researcher with information about the local environment of the nucleus in question and its motional properties. NMR spectroscopy is therefore often the preferred tool to elucidate molecular structure, flexibility, and interactions since NMR provides descriptions of the molecular dynamics and interactions at atomic resolution. The main aim of the proposed research is to explore and develop a relative avenue in biomolecular NMR spectroscopy, that it, carbon-detected NMR spectroscopy. Our new applications will allow us to, among others, visualise the motions and mechanisms of macromolecular machines.Within the research areas of protein complexes, macromolecular machines and intrinsically disordered proteins the main body of NMR spectroscopic applications rely on detection of proton magnetisations. This is because the magnetic strength of the proton is four times stronger than that of carbon and ten times stronger than that of nitrogen, thereby giving raise to stronger NMR signals that are easier to detect. However, the spin-physics and the nuclear interactions that govern the linewidth of the NMR signals also depend on the magnetic strength of the interacting nuclei and researchers have therefore been diluting the protons out in molecular machines to sharpen the NMR signals, avoid overlaps of signals, and thereby facilitate new characterisations.Others and the applicants of this proposal have recently shown the potential in using carbon-detected NMR spectroscopy for the investigation of large proteins and also intrinsically disordered proteins. Although the carbon-detected NMR signals are weaker than the proton detected signals there are many benefits that we are planning on exploiting and developing further with the new equipment that we are applying for. Firstly, carbon signals are in general sharper than the corresponding proton signals - because of their lower magnetic strength. Secondly, the average distance between carbon signals are much higher than that of proton signals, thereby leading to significantly less overlap of signals and facilitating analyses of the underlying parameters. In general terms, this means that we will be able to monitor motions and interactions of proteins during the formations of their structure (protein folding), macromolecular machines such as the cells protein producing machinery (Ribosome) and DNA remodelling enzymes (histone deacetylases). We all have excellent track-records in applying and developing new methodologies for NMR spectroscopy also within the field of carbon-detected NMR.The research will be carried out at the Institute of Structural and Molecular Biology (ISMB) at University College London (UCL). A state-of-the-art and stimulating research environment with dedicated NMR machines that are optimised for detection of carbon nuclei, and the highly collaborative environment and world-class expertise open up the possibility for many fruitful collaborations across disciplines and many novel applications to come.