A Novel Crosslinking Strategy for MS Structural Biology

MS 结构生物学的新型交联策略

• 批准号: BB/M001563/1
• 负责人: Joseph Gray
• 金额: $ 14.85万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM001563%2F1
• 关键词: Novel; Crosslinking; Strategy; MS; Structural

Proteins may be thought of as the essential 'factories' within living cells. They are intricate and complex in construction, and carry out a variety of functions essential for the existence of life. Understanding the inner workings of these factories requires knowledge of how they are assembled, both at the atomic level and at the level of higher-order multi-complex protein entities that perform essential functions, such as respiration and cell replication. Protein factories are dynamic structures, responding to input signals through interactions with other molecules, most frequently other proteins. Knowing what happens during the processing of signals and how output responses are generated relies on knowledge of both the protein 3D structure and any dynamic changes that take place to it at the molecular-level during such events.A deeper understanding of how one protein recognises and binds to another protein in order to regulate its function underpins most of the biological activity in living cells and is therefore crucial to how we may go about designing new molecules to prevent or enhance such interactions. Any advancement in our understanding of protein-protein interactions has the potential to widen the number of targets for modifying biological function by the use of drugs. Such knowledge may help to accelerate the development of new molecular medicines to treat most of our major serious diseases, as these often manifest themselves through protein interaction pathways. Structural biology is concerned with the study of protein 'shape' and how alterations in shape affect function. Structures ranging from individual proteins to large multicomponent cellular assemblies are studied. This challenging problem requires the integration of many different biophysical and biochemical protein analysis techniques. One comparatively recent technique to be applied to the problem of determining the spatial relationships between proteins in a complex or in close proximity through a binding event is the use of chemical cross-linking followed mass spectrometry analysis (XL-MS).Chemical cross-linking seeks to 'freeze' the 3D arrangement of protein chains in a complex by tethering them together using a reagent that forms a strong covalent link between adjacent regions, provided they fall within the distance of the span of the cross-linker. The covalently-linked regions of the proximal protein chains can then be excised from the protein backbone using an enzyme and the cross-linked complex carrying residues of both parent proteins analysed using mass spectrometry (MS). Modern MS can break these linked fragments into smaller pieces and determine their constituent amino acid residues. Using clever software algorithms we can decipher not only the identity of the two linked proteins that were in close proximity, but also ID the actual sites of linkage. This increases the resolution of this method for structure determination from the level of large protein domains down to even smaller sections, dubbed ''peptide-level resolution''. Better resolution in turn permits us to build better, more accurate models of the structures of multi-protein complexes.Unfortunately XL-MS in its present format exhibits a number of weaknesses. Current cross-linkers in general use are non-cleavable. This gives rise to large linked complexes upon excision that are tricky to analyse by MS due to their size and complex fragmentation patterns. Also XL-MS reagents tend not to react efficiently, making the linked peptides difficult to detect in protein digests.To tackle these current limiting issues we propose the introduction of an entirely new innovative cross-linking and MS analysis strategy that involves developing a novel class of cross-linking reagents which will allow detection of the linked peptides combined with simple automated MS data analysis.

蛋白质可以被认为是活细胞中必不可少的“工厂”。它们在结构上错综复杂，执行着生命存在所必需的各种功能。了解这些工厂的内部运作需要了解它们是如何组装的，无论是在原子水平上，还是在执行呼吸和细胞复制等基本功能的高阶多复杂蛋白质实体的水平上都是如此。蛋白质工厂是动态的结构，通过与其他分子，最常见的是其他蛋白质的相互作用来响应输入信号。要知道在信号处理过程中发生了什么，以及如何产生输出响应，取决于对蛋白质3D结构的了解，以及在此类事件期间在分子水平上对蛋白质发生的任何动态变化的了解。更深入地了解一种蛋白质如何识别并结合另一种蛋白质，以调节其功能，是活细胞中大多数生物学活动的基础，因此对于我们如何设计新的分子来防止或增强这种相互作用至关重要。我们在理解蛋白质-蛋白质相互作用方面的任何进步都有可能扩大通过使用药物来改变生物功能的靶点的数量。这些知识可能有助于加速开发新的分子药物来治疗我们大多数严重的疾病，因为这些药物通常通过蛋白质相互作用的途径表现出来。结构生物学是研究蛋白质“形状”以及形状变化如何影响功能的学科。研究的结构范围从单个蛋白质到大型多组分细胞组件。这一具有挑战性的问题需要许多不同的生物物理和生化蛋白质分析技术的集成。一种相对较新的技术被应用于确定蛋白质在复合体中或通过结合事件接近的蛋白质之间的空间关系的问题是使用化学交联物随后的质谱分析(XL-MS)。化学交联物寻求通过使用在交联剂跨度范围内形成强共价键的试剂将蛋白质链在复合体中的3D排列固定在一起，从而将蛋白质链‘冻结’在复合体中的三维排列。然后，可以使用一种酶将近端蛋白质链的共价连接区域从蛋白质主干上移除，并使用质谱仪(MS)分析携带两种亲本蛋白质残基的交联复合体。现代MS可以将这些连接的片段分解成更小的片段，并确定它们的组成氨基酸残基。使用巧妙的软件算法，我们不仅可以破译两个紧密相连的蛋白质的身份，还可以识别出实际的连锁位点。这提高了这种确定结构的方法的分辨率，从大的蛋白质结构域水平向下到甚至更小的部分，被称为“肽水平分辨率”。更好的分辨率反过来又允许我们建立更好、更准确的多蛋白质复合体结构模型。不幸的是，目前格式的XL-MS显示出一些弱点。目前普遍使用的交联剂是不可切割的。这会在切除后产生大的连接复合体，由于它们的大小和复杂的碎片模式，MS很难分析它们。此外，XL-MS试剂往往反应效率低下，使得连接的多肽很难在蛋白质消化中被检测到。为了解决这些目前的限制问题，我们建议引入一种全新的创新的交联和MS分析策略，包括开发一种新型的交联试剂，它将允许检测连接的多肽与简单的自动化MS数据分析相结合。

项目成果

Traceless Cleavage of Protein-Biotin Conjugates under Biologically Compatible Conditions.

• DOI: 10.1002/cbic.201700214
• 发表时间: 2017-09-05
• 期刊: Chembiochem : a European journal of chemical biology
• 作者: Cowell J;Buck M;Essa AH;Clarke R;Vollmer W;Vollmer D;Hilkens CM;Isaacs JD;Hall MJ;Gray J
• 通讯作者: Gray J

A Novel crosslinking Strategy for MS Structural Biology.

MS 结构生物学的新型交联策略。

• 发表时间: 2015
• 作者: Murray J