A Biological Pulsed EPR/ENDOR Facility for the Manchester Interdisciplinary Biocentre

曼彻斯特跨学科生物中心的生物脉冲 EPR/ENDOR 设施

• 批准号: BB/E013007/1
• 负责人: Stephen Rigby
• 金额: $ 30.58万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE013007%2F1
• 关键词: Biological; Pulsed; EPR; ENDOR; Facility

Proteins are dynamic, complex structures that facilitate cellular communication, catalysis, structure, growth and division through their interaction with other biological macromolecules, enzyme substrates and ligands. Biophysical methods are crucial for determining the structure and function of protein molecules. EPR spectroscopy has emerged as a major spectroscopic technique for the structural characterization of protein complexes, membrane protein systems, analysis of protein dynamics and the chemistry catalysed by enzymes. Modern pulsed EPR methods provide important information on protein structure through triangulation of engineered spin labels or natural 'spin active' cofactors present in proteins. EPR spectroscopy can also provide detailed electronic structural information about reactive centres (cofactors and protein based radicals) present in enzyme catalysts, and time-resolved information about the chemistry catalysed by protein systems. The contents of cells are protected and enclosed by an outer sheath or membrane composed of proteins as well as fat molecules (lipids) that form a relatively impermeable barrier. Such membranes are also found inside the cell, and form compartments that have specialised functions. The proteins found in membranes often act as gatekeepers, allowing, or sometimes actively pumping, molecules through the membrane. They also have a range of other functions such as enzymes, sensors (e.g. of hormones) and as scaffolding to provide structural support. Structural information for membrane-bound systems is scarce owing to the difficulties of applying traditional structural approaches (e.g. crystallography) to membrane systems. Spin label EPR spectroscopy provides valuable distance information from which the fold/structure of membrane proteins can be investigated. Unravelling the structure of membrane proteins is one of the major research themes in Manchester, and solid-state NMR, X-ray crystallography and cryo-electron microscopy are all employed to extract structural data. Work in this area would be significantly enhanced by the provision of EPR facilities to measure distance relationships and conformational dynamics. The Manchester group forms one node of the membrane protein structure initiative, a structural proteomics initiative sponsored by the UK research council BBSRC. RNA, in its varied forms, interacts with protein to carry out fundamental roles in the cell. Understanding the contributions of various RNAs to the control of translation in the cell forms an important theme within the structural biology and biophysics group. The understanding of molecular recognition events, including those involved in assembly of macromolecular complexes consisting of both protein and RNA are a challenge for biochemical, biophysical and structural study. The extracellular matrix group forms one of the major research centres at Manchester. The enormous size of extracellular matrix complexes, such as collagen fibrils, necessitates the use of novel structural methods. EPR spectroscopy is ideal in this regard by providing distance relationships in large protein complexes. By combining the lower resolution data from these studies, with higher resolution data for protein components, or fragments of the fibrils, a picture of the architecture of these cellular structures will emerge. Finally, catalysts in biology have properties that chemists would love to emulate. Biological reactions have exquisite specificity, even down to generating a single stereoisomer, and also do not need high temperatures and pressures. To fully understand how these processes are achieved in biology, structural biology must provide atomic structures of the protein catalysts and details (at the quantum level) of reaction mechanism. Manchester has a large grouping in this area and modern EPR facilities will provide much needed electronic structure and time-resolved information to established programmes in this area of biocatalysis.

蛋白质是动态的、复杂的结构，通过它们与其他生物大分子、酶底物和配体的相互作用，促进细胞通信、催化、结构、生长和分裂。生物物理方法对于确定蛋白质分子的结构和功能至关重要。电子顺磁共振波谱已成为表征蛋白质复合体结构、膜蛋白体系、蛋白质动力学分析和酶催化化学的主要波谱技术。现代脉冲EPR方法通过对存在于蛋白质中的工程自旋标记或天然“自旋活性”辅因子进行三角测量，提供了关于蛋白质结构的重要信息。EPR波谱还可以提供关于酶催化剂中存在的反应中心(辅因子和基于蛋白质的自由基)的详细电子结构信息，以及关于蛋白质体系催化的化学的时间分辨信息。细胞的内容物被由蛋白质和脂肪分子(脂)组成的外鞘或膜保护和包裹，形成相对不透水的屏障。这种膜也存在于细胞内，形成具有特殊功能的隔室。膜中发现的蛋白质通常充当守门人，允许分子通过膜，有时甚至是主动泵入分子。它们还具有一系列其他功能，如酶、传感器(如激素)和作为脚手架提供结构支持。由于将传统的结构方法(例如结晶学)应用于膜系统的困难，膜结合系统的结构信息很少。自旋标记EPR谱提供了有价值的距离信息，从这些信息可以研究膜蛋白的折叠/结构。解开膜蛋白的结构是曼彻斯特的主要研究主题之一，固体核磁共振、X射线结晶学和冷冻电子显微镜都被用来提取结构数据。通过提供EPR设施来衡量距离关系和构象动态，这一领域的工作将得到显着加强。曼彻斯特小组组成了膜蛋白结构倡议的一个节点，该倡议是由英国研究委员会BBSRC发起的结构蛋白质组倡议。RNA以各种形式与蛋白质相互作用，在细胞中发挥基本作用。了解各种RNA对细胞内翻译控制的贡献形成了结构生物学和生物物理学小组的一个重要主题。理解分子识别事件，包括那些涉及蛋白质和RNA组成的大分子复合体组装的分子识别事件，是生化、生物物理和结构研究的一个挑战。细胞外基质小组构成了曼彻斯特的主要研究中心之一。细胞外基质复合体的巨大尺寸，如胶原纤维，需要使用新的结构方法。EPR光谱在这方面是理想的，因为它提供了大型蛋白质复合体的距离关系。通过将这些研究的较低分辨率数据与蛋白质成分或纤维碎片的较高分辨率数据相结合，这些细胞结构的结构图景将浮出水面。最后，生物学中的催化剂具有化学家们乐于效仿的特性。生物反应具有极高的专一性，甚至可以生成单一的立体异构体，而且也不需要高温和压力。为了充分了解这些过程是如何在生物学中实现的，结构生物学必须提供蛋白质催化剂的原子结构和反应机理的细节(在量子水平上)。曼彻斯特在这一领域有一个庞大的群体，现代的EPR设施将为这一生物催化领域的现有方案提供急需的电子结构和时间分辨信息。

项目成果

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1.

结构表征和配体/抑制剂鉴定提供了对结核分枝杆菌细胞色素P450 CYP126A1的功能见解。

• DOI: 10.1074/jbc.m116.748822
• 发表时间: 2017-01-27
• 期刊: The Journal of biological chemistry
• 作者: Chenge JT;Duyet LV;Swami S;McLean KJ;Kavanagh ME;Coyne AG;Rigby SE;Cheesman MR;Girvan HM;Levy CW;Rupp B;von Kries JP;Abell C;Leys D;Munro AW
• 通讯作者: Munro AW

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

• DOI: 10.1038/srep26628
• 发表时间: 2016-05-26
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Chenge J;Kavanagh ME;Driscoll MD;McLean KJ;Young DB;Cortes T;Matak-Vinkovic D;Levy CW;Rigby SE;Leys D;Abell C;Munro AW
• 通讯作者: Munro AW