Probing biomolecular interactions by combining ETD-tandem mass spectrometry with chemical footprinting methodologies.

通过将 ETD 串联质谱法与化学足迹方法相结合来探测生物分子相互作用。

• 批准号: BB/M012573/1
• 负责人: Alison Ashcroft
• 金额: $ 43.47万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM012573%2F1
• 关键词: Probing; biomolecular; interactions; combining; ETD

In life, most proteins function as an integral part of a biomolecular complex consisting of proteins, nucleic acids, small molecules and metal ions, all held together in a specific 3D architecture. Correctly functioning proteins maintain health in plants and animals, the latter including humans. However, protein mis-function can lead to disease: for example, unwanted protein self-aggregation can produce insoluble amyloid plaques which are associated with well-known diseases such as Alzheimer's, Parkinson's and Type II diabetes. We propose to purchase a state-of-the-art mass spectrometer which will enable us to pin-point protein-protein and protein-ligand binding sites. Our strategy involves subjecting a protein, or protein complex, which is in its native (3D) conformation, to chemical modification (or "footprinting") techniques. The regions of the protein that will be modified chemically are those which are exposed on the exterior of its structure, whilst the regions lying in the interior of the structure are inaccessible and remain unmodified.In order to identify the sites of chemical modification, we need to use advanced mass spectrometric techniques that will tell us not only the molecular mass of the protein, but also its amino acid sequence. During protein sequencing, it is apparent which amino acids have been modified due to the change in the mass of the residue due to the chemical modification. For example, if the amino acid is oxidised, it will gain an oxygen atom and a corresponding increase of 16 Da. Our proposed combination of advanced mass spectrometry with chemical modification methods will provide new information concerning: a. the 3D structure of the protein by disclosing which regions are on the surface and which are in the interior of the structure; b. protein-protein and protein-ligand binding sites, by comparing the "footprint" of the protein alone with that of the protein bound to a small molecule or another protein, we can discern where the ligand is bound; c. protein function. We can monitor the protein's chemical footprint over time to map any structural changes occurring e.g. unfolding, binding, self-aggregation. This will provide key insights into how proteins function and mis-function; d. the design of potential therapeutics. To prevent a protein from mis-functioning, it is often possible to add a small molecule drug to prevent an unwanted event e.g. to prevent a protein from unfolding followed by self-aggregation, it is possible to add a small molecule which will compete successfully for the protein-protein binding site. Our methods will locate the binding site.The beneficiaries of these proposed studies will be the bio-scientists who will gain a significantly improved understanding of the ways in which proteins and other biomolecules function and mis-function. This will lead to improved drug design by pharmaceutical and biopharma companies, which in turn will lead to a healthier life for all of us.

在生活中，大多数蛋白质作为生物分子复合物的组成部分，生物分子复合物由蛋白质，核酸，小分子和金属离子组成，所有这些都在特定的3D结构中保持在一起。正常运作的蛋白质维持植物和动物的健康，后者包括人类。然而，蛋白质功能失调可能导致疾病：例如，不需要的蛋白质自聚集可能产生不溶性淀粉样蛋白斑块，其与众所周知的疾病如阿尔茨海默氏症、帕金森氏症和II型糖尿病相关。我们建议购买一台最先进的质谱仪，这将使我们能够精确定位蛋白质与蛋白质和蛋白质与配体的结合位点。我们的策略包括对天然（3D）构象的蛋白质或蛋白质复合物进行化学修饰（或“足迹”）技术。蛋白质的化学修饰区域是暴露在其结构外部的区域，而位于结构内部的区域是不可接近的，保持未修饰状态。为了确定化学修饰的位点，我们需要使用先进的质谱技术，不仅可以告诉我们蛋白质的分子量，还可以告诉我们其氨基酸序列。在蛋白质测序过程中，由于化学修饰引起的残基质量变化，哪些氨基酸被修饰是显而易见的。例如，如果氨基酸被氧化，它将获得一个氧原子，并相应增加16 Da。我们提出的先进质谱与化学修饰方法的组合将提供有关以下方面的新信息：a.通过公开哪些区域在所述结构的表面上以及哪些区域在所述结构的内部来描述所述蛋白质的3D结构; B.蛋白质-蛋白质和蛋白质-配体结合位点，通过将单独的蛋白质的“足迹”与结合到小分子或另一种蛋白质的蛋白质的“足迹”进行比较，我们可以辨别配体结合的位置; c.蛋白质功能我们可以监测蛋白质随时间的化学足迹，以绘制发生的任何结构变化，例如解折叠，结合，自聚集。这将为蛋白质如何发挥功能和错误功能提供关键见解; d.潜在疗法的设计。为了防止蛋白质功能失调，通常可以添加小分子药物以防止不想要的事件，例如为了防止蛋白质解折叠随后自聚集，可以添加将成功竞争蛋白质-蛋白质结合位点的小分子。我们的方法将定位结合位点。这些拟议研究的受益者将是生物科学家，他们将获得对蛋白质和其他生物分子功能和错误功能的方式的显着改进的理解。这将导致制药和生物制药公司改进药物设计，从而为我们所有人带来更健康的生活。

项目成果

Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

BH3 配体对 MCL-1 的光催化邻近标记

• DOI: 10.26434/chemrxiv.7862006.v1
• 发表时间: 2019
• 作者: Beard H

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane.

• DOI: 10.1038/s41467-021-23146-4
• 发表时间: 2021-05-14
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Byrne MJ;Iadanza MG;Perez MA;Maskell DP;George RM;Hesketh EL;Beales PA;Zack MD;Berry C;Thompson RF
• 通讯作者: Thompson RF

Photocatalytic proximity labelling of MCL-1 by a BH3 ligand.

• DOI: 10.1038/s42004-019-0235-z
• 发表时间: 2019-11-21
• 期刊: Communications chemistry
• 影响因子: 5.9
• 作者: Beard HA;Hauser JR;Walko M;George RM;Wilson AJ;Bon RS
• 通讯作者: Bon RS

Structural basis of rotavirus RNA chaperone displacement and RNA annealing.

• DOI: 10.1073/pnas.2100198118
• 发表时间: 2021-10-12
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Bravo JPK;Bartnik K;Venditti L;Acker J;Gail EH;Colyer A;Davidovich C;Lamb DC;Tuma R;Calabrese AN;Borodavka A
• 通讯作者: Borodavka A

A bovine antibody possessing an ultralong complementarity-determining region CDRH3 targets a highly conserved epitope in sarbecovirus spike proteins.

• DOI: 10.1016/j.jbc.2022.102624
• 发表时间: 2022-12
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Burke, Matthew J.;Scott, James N. F.;Minshull, Thomas C.;Gao, Zeqian;Manfield, Iain;Savic, Sinisa;Stockley, Peter G.;Calabrese, Antonio N.;Boyes, Joan
• 通讯作者: Boyes, Joan