High Sensitivity Cryoprobe Equipment for the NMR Facility of the Department of Biochemistry University of Cambridge

核磁共振波谱学已成为研究生物大分子（如蛋白质和核酸）在溶液中的自然环境中的结构和迁移率的有力工具。NMR的一个特别强大的特征是可以在原子水平上研究分子的相互作用，这允许区分蛋白质中的功能区域和结构区域，并允许表征哪些残基参与分子水平上的相互作用。可以确定这种相互作用的强度和时间尺度，这揭示了这种相互作用背后的潜在物理机制的信息。不幸的是，NMR光谱是一种相对不敏感的方法，因此目前大多数可用的研究仅限于< 20 kDa的较小蛋白质。最近，新的技术和改进的光谱仪硬件导致了基本灵敏度的急剧增加，从而使更大的生物大分子的研究成为可能。其中一个重大进展是引入了所谓的冷冻探针，将NMR实验的灵敏度提高了2到5倍。这种显著更高的灵敏度可以用于良好的优势，以研究较大的蛋白质及其与通常无法用常规设备获得的其他蛋白质或DNA/RNA的复合物，以研究丰度较低或聚集的蛋白质，或减少进行特定NMR实验所需的时间，从而增加光谱仪的容量。我们的部门NMR设施形成了一个协作研究环境，为来自生物化学和化学部门的广泛用户提供服务，这些用户从事多种结构项目。这些项目中的许多涉及大蛋白质或多蛋白质复合物的工作，或者必须在低蛋白质浓度下进行，使得这项工作将从这种冷冻探针设备的可用性中显著受益。在这项申请中，我们要求资金在我们四台设备中的两台上安装冷冻探针，以便能够继续我们提出的研究项目。我们概述的一些项目要求极高，高灵敏度NMR设备是其可行性的先决条件。以下是从我们的研究计划中选择的一些生物学和临床重要性的例子。我们的工作领域包括涉及细胞信号传导许多方面的分子和复合物的结构研究及其在细胞运动和癌症中的意义，以及涉及例如范可尼贫血（一种导致进行性骨髓衰竭的疾病）的癌症易感综合征蛋白的研究。进一步的项目研究蛋白质错误折叠的不同方面，例如可以在患有阿尔茨海默氏症或帕金森氏症的衰弱性疾病的患者的脑组织中的斑块中发现。对染色质的研究是另一个重要的研究领域，染色质是控制基因表达的中心，它与DNA和许多蛋白质的相互作用。对完整核糖体（蛋白质合成发生的细胞中心）的结构研究相对较新，并且由于附着于核糖体的合成新生蛋白质的浓度非常低且寿命有限而特别具有挑战性。作为最后一个例子，我们提到膜蛋白的结构研究。这些蛋白质占CA。它占所有编码蛋白质的30%，并在分子水平上控制着广泛的生物功能，如呼吸，信号传导和运输。由于它们是水不溶性的，因此很难获得结构信息。NMR可以研究这种蛋白质的洗涤剂溶解形式，但这种复合物的大尺寸需要最高的NMR灵敏度。

NMR spectroscopy has emerged as a powerful tool to study the structure and mobility of biomacromolecules such as proteins and nucleic acids in their natural environment in solution. A particularly powerful feature of NMR is that interactions of molecules can be studied at an atomic level that allows to differentiate e.g. functional from structural regions in a protein and permit to characterize which residues are involved in a interaction at molecular level. Strengths and timescales of such interactions can be determined which reveal information about the underlying physical mechanisms behind such interactions. Unfortunately, NMR spectroscopy is a relatively insensitive method so that the majority of currently available studies have been limited to smaller proteins < 20 kDa. More recently novel techniques and improved spectrometer hardware have led to a dramatic increase in the basic sensitivity so that the study of larger biomacromolecules has become possible. One such significant advance has been the introduction of the so-called cryoprobe that improves the sensitivity of NMR experiments between two and five-fold. This dramatically higher sensitivity can be used to good advantage, to study larger proteins and their complexes with other proteins or DNA/RNA typically not accessible with conventional equipment, to investigate less abundant or aggregating proteins, or to reduce the time required to perform a particular NMR experiment, thus increasing the capacity of the spectrometer. Our Departmental NMR facility forms a collaborative research environment that serves a wide range of users from the Departments of Biochemistry and Chemistry that work on a multitude of structural projects. Many of these projects involve work with large proteins, or multi-protein complexes or have to be conducted at low protein concentrations so that this work would dramatically benefit from the availability of such cryoprobe equipment. In this application we are requesting funds to install cryoprobes on two out of our four facility machines to be able to pursue our proposed research projects. Some of our outlined projects are extremely demanding and high sensitivity NMR equipment is a pre-requisite for their feasibility. Here are some selected examples of biological and clinical importance from our research plan. Our areas of work cover structural studies of molecules and complexes involved in many aspects of cell-signalling and their implications in cell motility and cancer and the study of cancer susceptibility syndrome proteins involved e.g. in Fanconi Anaemia, a disease that leads to progressive bone marrow failure. Further projects study different aspects of misfolding of proteins such as can be found in plaque in brain tissue of patients that have the debilitating diseases of Alzheimer's or Parkinson's. The study of chromatin, central to the control of gene expression, it's interactions with DNA and many proteins is another major significant research area. Structural studies on intact ribosomes, the cellular centers where protein synthesis occurs, are relatively new and particularly challenging to perform due to the very low concentrations and limited lifetimes of the synthesized nascent protein attached to the ribosome. As a final example we mention the structural study of membrane proteins. These proteins account for ca. 30% of all encoded proteins and control a vast range of biological functions such as respiration, signalling and transport at a molecular level. As they are water insoluble structural information is hardly available. NMR can study such proteins in a detergent solubilized form but the large size of such complexes requires the highest NMR sensitivity available.