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，对许多重要过程至关重要，从蛋白质折叠和展开，配体结合，信号传导，变构调节到酶催化。因此，在分子水平上理解运动提供了有价值的见解，涉及结构变化的现象，无论是当他们作为预期的功能或当他们故障。例如，了解蛋白质如何错误折叠可能有助于对抗称为淀粉样变性的衰弱性疾病，包括阿尔茨海默病，II型糖尿病或牛海绵状脑病，更广泛地称为“疯牛病”。此外，了解与信号通路内在相关的运动可能会导致开发出更好的针对这些通路的药物（大多数药物都是这样工作的）。甚至开发实用的环境友好的生物电池和生物燃料电池也可以通过了解它们利用酶时的分子运动来帮助。因此，设计出以原子分辨率测量蛋白质运动的方法非常重要。为此，本课题将开发利用原子核固有磁性的核磁共振（NMR）技术。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施加特定频率的无线电波来改变。通过测量相关的频率，可以了解原子相对于彼此的相对位置以及该位置如何随时间变化，即分子运动。这种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。在固态NMR中，这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，其涉及样品围绕与外部磁场成54.7度角倾斜的轴的快速旋转。最近推出的尖端仪器可以实现高达每秒100 000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 kHz MAS的探头。MAS在如此惊人的频率下的效率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的位点特异性1H弛豫或高度灵敏的1H检测弛豫测量。100 kHz的旋转消除了许多不希望的影响，模糊了分子运动的参数报告的测量，从而允许获得蛋白质运动的详细视图。国家核磁共振光谱方法，利用新的100千赫自旋制度，并将提供更好的访问，以测量蛋白质的动态过程中，原子解决方案，并以特定的方式。特别是，我们将专注于技术，提供访问慢动作的制度，是难以访问的固态NMR的姐妹方法-溶液NMR。此外，为了提高开发技术的实用性，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.