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，对蛋白质折叠和展开、配体结合、信号传导、变构调节和酶催化等许多重要过程至关重要。因此，在分子水平上理解运动提供了对涉及结构变化的现象的有价值的见解，无论是当它们正常工作时还是当它们发生故障时。例如，了解蛋白质如何错误折叠可能有助于对抗淀粉样变性疾病，包括阿尔茨海默病、2型糖尿病或俗称“疯牛病”的牛海绵状脑病。此外，了解与信号通路内在相关的运动可能会导致开发针对这些通路的更好的药物（大多数药物都是这样工作的）。即使是开发实用的环境友好型生物电池和生物燃料电池，也可能得益于分子运动的知识，因为它们利用了酶。因此，设计出在原子分辨率上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，我们将开发一种叫做核磁共振（NMR）的技术，它依赖于原子核的固有磁性。当放置在强磁场中时，原子核的磁矩与外部磁场排列一致，但这种排列可以通过特定频率的无线电波的应用而改变。通过测量相关频率，人们可以了解原子相对于彼此的相对位置，以及这个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，人们不仅可以了解整个分子的信息，还可以了解其中特定原子的信息。固态核磁共振是该项目中使用的主要方法，通过一种称为魔角旋转（MAS）的技术，可以实现区分单个位点所需的高分辨率，该技术涉及样品围绕与外部磁场倾斜54.7度的轴快速旋转。最近引进的尖端仪器可以实现每秒10万转的旋转频率。该项目的中心是购买英国第一个能够达到100千赫MAS的探测器。在如此惊人的频率下，MAS效率的提高使得设计新的实验成为可能，这些实验提供了新的分析工具来访问运动，例如，在完全质子化的样品中，特定位点的1H弛豫或高灵敏度的1H检测弛豫测量。100千赫旋转消除了一些不希望的影响，使分子运动参数报告的测量模糊不清，从而可以获得蛋白质运动的详细视图。在这个项目中，我们建议开发一系列强大的固态核磁共振光谱方法，利用新的100千赫旋转机制，并将提供改进的途径，以原子分辨率和特定位点的方式测量蛋白质的动态过程。特别是，我们将重点关注那些在固态核磁共振姐妹方法-溶液核磁共振难以获得的状态下提供慢运动的技术。此外，为了提高所开发技术的实用性，我们将优化它们的速度和灵敏度。

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.