A Novel Crosslinking Strategy for MS Structural Biology

蛋白质可以被认为是活细胞内必不可少的“工厂”。它们的结构错综复杂，执行着生命存在所必需的各种功能。要了解这些工厂的内部工作原理，就需要了解它们是如何在原子水平和执行基本功能（如呼吸和细胞复制）的高阶多复杂蛋白质实体的水平上组装的。蛋白质工厂是动态结构，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，以及输出响应是如何产生的，需要了解蛋白质的3D结构，以及在这些事件中在分子水平上发生的任何动态变化。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活动的基础，因此对于我们如何设计新分子来防止或增强这种相互作用至关重要。我们对蛋白质-蛋白质相互作用的理解的任何进步都有可能通过使用药物来改变生物功能的靶标数量。这些知识可能有助于加速新分子药物的开发，以治疗我们的大多数重大严重疾病，因为这些疾病通常通过蛋白质相互作用途径表现出来。结构生物学关注的是蛋白质“形状”的研究以及形状的改变如何影响功能。结构范围从单个蛋白质到大的多组分细胞组件的研究。这个具有挑战性的问题需要整合许多不同的生物物理和生化蛋白质分析技术。一种相对较新的技术被应用于确定一个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质谱分析（xml - ms）。化学交联试图“冻结”复合体中蛋白质链的3D排列，方法是使用一种试剂将它们拴在一起，在相邻区域之间形成强共价键，前提是它们位于交联剂的范围内。然后可以使用一种酶从蛋白质主干上切除近端蛋白质链的共价连接区域，并使用质谱（MS）分析携带亲本蛋白残基的交联复合物。现代质谱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。使用聪明的软件算法，我们不仅可以破译两个相邻的连接蛋白的身份，还可以识别连接的实际位置。这增加了该方法的结构测定的分辨率，从大的蛋白质结构域水平下降到更小的部分，被称为“肽水平分辨率”。更高的分辨率反过来使我们能够建立更好、更精确的多蛋白复合物结构模型。不幸的是，目前格式的xml - ms显示出许多弱点。目前一般使用的交联剂是不可切割的。这在切除后产生了大的连接复合物，由于它们的大小和复杂的碎片模式，用质谱分析是棘手的。此外，xml - ms试剂往往不能有效地反应，使得连接肽难以在蛋白质消化中检测到。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交联和质谱分析策略，包括开发一类新型的交联试剂，该试剂将允许检测连接肽并结合简单的自动化质谱数据分析。

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.