100 kHz magic angle spinning for development of solid-state NMR methodology for probing protein dynamics

运动和变化是生物体的基本特征，对从蛋白质折叠和解折叠、配体结合、信号传递、变构调节到酶催化等许多生命过程都是至关重要的。因此，在分子水平上理解运动提供了对结构变化现象的有价值的见解，无论是当它们发挥预期的功能时，还是当它们发生故障时。例如，了解蛋白质的错误折叠可能有助于对抗被称为淀粉样变性的衰弱疾病，这些疾病包括阿尔茨海默氏症、II型糖尿病或牛海绵状脑病，更广为人知的是，这种疾病被称为“疯牛病”。此外，了解与信号通路有内在联系的运动可能会导致针对这些通路的更好药物的开发(大多数药物都是这样工作的)。即使是实用的环境友好型生物制药和生物燃料电池的开发，也可能得到分子运动知识的帮助，因为它们利用酶。因此，设计出以原子分辨率测量蛋白质运动的方法是非常重要的。为了做到这一点，在这个项目中，我们将开发一种名为核磁共振(NMR)的技术，它依赖于原子核的固有磁性。当置于强磁场中时，原子核的磁矩与外部磁场对齐，但这种对齐可以通过应用特定频率的无线电波来改变。通过测量相关频率，人们可以了解原子之间的相对位置，以及这种位置如何随时间变化，即分子运动。这项技术的一个非常强大的方面是，人们不仅可以了解一个分子的整体信息，而且可以了解其中特定原子的信息。在固态核磁共振中，这是该项目中使用的主要方法，通过一种名为魔角旋转(MAS)的技术实现了区分各个位置所需的高分辨率，该技术涉及样品绕与外部磁场成54.7度角倾斜的轴快速旋转。最近推出的尖端仪器可以实现高达每秒10万转的旋转频率。该项目的中心是购买英国第一个能够进行100 kHzMAS的探测器。MAS在如此惊人的频率下效率的提高使设计新的实验成为可能，这些实验提供了访问运动的新分析工具，例如在完全质子化的样品中进行特定位置的1H弛豫或高灵敏度的1H弛豫测量。在这个项目中，我们建议开发一系列健壮的固态核磁共振波谱方法，这些方法利用新的100 kHz旋转系统，并将提供更好的途径，以原子分辨率和特定位置的方式测量蛋白质的动态过程。特别是，我们将重点介绍在固态核磁共振姊妹方法--溶液核磁共振难以访问的区域中提供访问慢动作的技术。此外，为了提高所开发技术的实用性，我们将在速度和灵敏度方面对其进行优化。

Motion and change are essential features of living organisms and fundamentally important for many vital processes from protein folding and unfolding, ligand binding, signalling, allosteric regulation to enzymatic catalysis. Consequently, understanding motions at molecular level provides valuable insights into the phenomena involving change of structure both when they function as intended or when they malfunction. For example understanding how proteins misfold may help to fight debilitating diseases called amyloidoses that include Alzheimer's disease, type II diabetes or bovine spongiform encephalopathy more widely known as "mad cow" disease. Moreover, understanding motions that are intrinsically associated with signalling pathways may result in development of better drugs that target such pathways (most medicines work this way). Even development of practical environmentally friendly biobatteries and biofuel cells may be aided by knowledge of molecular motions as they make use of enzymes. Thus it is really important to devise ways to measure protein motions at atomic resolution.To do that, in this project, we will develop a technique called nuclear magnetic resonance (NMR), which relies on the inherent magnetism of atomic nuclei. When placed in a strong magnetic field magnetic moments of nuclei align with the external field but this alignment may be changed by application of radio waves at specific frequencies. By measuring the associated frequencies one can learn about the relative position of atoms with respect to each other and how this position changes with time i.e. molecular motions. A very powerful aspect of this technique is that one can learn such information not only for a molecule overall but for specific atoms in it. In solid-state NMR, which is the primary method used in this project, the high resolution necessary to distinguish individual sites is enabled by a technique called magic angle spinning (MAS), which involves fast rotation of the sample around an axis inclined at an angle of 54.7 degrees to the external magnetic field. Recently introduced cutting edge instrumentation allows achieving spinning frequencies up to 100 000 revolutions per second. The centre of this project is the purchase of the first in the UK probe capable of 100 kHz MAS. The improved efficiency of MAS at such astounding frequencies makes possible designing new experiments that provide new analytical tools to access motions, e.g. site-specific 1H relaxation or highly sensitive 1H-detected relaxation measurements in fully protonated samples. The 100 kHz spinning removes a number of undesired effects obscuring the measurements of parameters reporting on molecular motions and thus allows a detailed view of protein motions to be obtained.In this project we propose to develop a series of robust solid-state NMR spectroscopic methods that take advantage of the new 100 kHz spinning regime and will provide improved access to measuring of dynamic processes in proteins at atomic resolution and in a site-specific manner. In particular, we will focus on techniques that provide access to slow motions in the regime that is difficult to access by the solid-state NMR sister method - solution NMR. In addition, in order to improve practicality of the developed techniques we will optimise them for speed and sensitivity.