Structural analyses of multicomponent protein complexes by analytical ultracentrifugation

分析超离心（AUC）是一种强大的方法，可以详细研究蛋白质在溶液中的结合或降解。将蛋白质样品插入顶部和底部有窗口的细胞组件中。细胞被放置在一个钛转子中，然后在分析型超离心机内以每分钟50000转的速度旋转。在高速或低速实验中，光学系统被用来观察蛋白质，在高速或低速实验中，蛋白质缓慢地移动到转子的外部，在它移动的过程中被连续观察。在实验期间记录了多达200-500次扫描。高速“速度”实验测量蛋白质移动到细胞底部的速度，从中我们了解蛋白质的形状，以及样品中存在多少不同的物种。这对于分析不同蛋白质之间形成的复合物，或发现样品中存在多少种不同类型的蛋白质，以及它们是相关的还是裂解的特别有用。低速“平衡”实验平衡了蛋白质在细胞内扩散的趋势和沉积到细胞底部的趋势。这些数据告诉我们溶液中蛋白质的大小和样品中任何结合行为的强度。现代AUC仪器提供了丰富的蛋白质新信息，可以使用新的强大的软件包破译。例如，所有的速度扫描都可以输入到SEDFIT等软件中，结果可以识别出溶液中存在的所有大分子物种，甚至是较小的物种。然后我们可以详细剖析蛋白质复合物的形成，包括确定它们形成的结合常数，或者在其他情况下跟踪蛋白质降解或切割。其他软件如SEDANAL或SEDPHAT详细分析平衡扫描。因此，现代AUC使新型实验成为可能，在这些实验中，蛋白质复合物可以作为许多重要的生物学变量（如辅因子和抑制剂）的功能进行研究，以阐明负责活性和功能的机制。所要求的AUC将应用于关键问题。在机体的补体免疫防御系统中，我们将分析补体激活的大量调节因子H与其靶标的多重相互作用。因子H的生物学特性很重要，因为它与失明和肾衰竭相关的炎症性疾病有关，因此控制其行为的能力具有很大的优势。抗体在免疫学中也很重要。我们可以使用AUC数据来更好地了解抗体识别入侵身体的外来物质的方式，以及抗体如何与细胞表面受体结合以控制免疫反应。酶在许多工业应用中都很重要，因此发现新的方法来制造更强大的酶来进行化学反应变得至关重要。AUC将帮助我们识别那些被重新设计得更稳定的酶。我们将使用AUC来研究称为TIP48和TIP49的特殊人类蛋白质如何相互关联以及如何被小分子修饰。这两种蛋白质都利用化学能在大型核复合物中发挥作用。寡聚化是至关重要的偶联ATP水解这些蛋白质的分子作用。将研究古细菌中热稳定形式的TIP49，以发现这些小分子在结合过程中的重要性，以及删除部分TIP49对其亚基组织的影响。一组不同的蛋白质参与有丝分裂，细胞分裂的过程。AUC对于确定这些有丝分裂复合体的形成方式及其稳定性将是无价的，这项工作对于通过其他方法确定更详细的分子结构至关重要。

Analytical ultracentrifugation (AUC) is a powerful method that enables protein association or degradation in solution to be studied in detail. Protein samples are inserted into a cell assembly with windows at the top and bottom. The cells are placed into a titanium rotor which is then spun at speeds up to 50,000 revs per min inside the analytical ultracentrifuge. Optical systems are used to observe the protein in either high-speed or low-speed experiments in which the protein slowly moves to the outside of the rotor, being continuously observed as it moves. Up to 200-500 scans are recorded during the experiment. The high speed 'velocity' experiments measure how quickly the protein moves to the bottom of the cell, from which we learn about the shape of the protein, and how many different species exist in the sample. This is especially useful for analysing complexes formed between different proteins, or discovering how many different types of proteins are present in the sample, and whether they are associated or cleaved. The low speed 'equilibrium' experiments balance the tendency of the protein to diffuse in the cell with that to sediment to the bottom of the cell. This data tells us about the size of the protein in solution and the strength of any associative behaviour in the sample. Modern AUC instrumentation provides a wealth of new information on proteins that can be deciphered using new powerful software packages. For example, all the velocity scans can be inputted into software such as SEDFIT, as the result of which all the macromolecular species present in the solution can be identified, even the minor ones. We can then dissect the formation of protein complexes in detail, including determining the association constants for their formation, or follow protein degradation or cleavage in other cases. Other software such as SEDANAL or SEDPHAT analyses equilibrium scans in detail. Hence the modern AUC makes possible new types of experiments in which protein complexes can be studied as a function of many biologically important variables such as cofactors and inhibitors in order to clarify the mechanisms responsible for activity and function. The requested AUC will be applied to key problems. In the complement immune defence system of the body, we will analyse the multiple interactions made by an abundant regulator of complement activation called Factor H with its targets. The biology of Factor H is important as this has been implicated in inflammatory disorders related to blindness and kidney failure, so the ability to control its behaviour has great advantages. Antibodies are also important in immunology. We can use AUC data to understand better the way in which antibodies recognise foreign material that invades the body and how antibodies bind to cell surface receptors to control the immune response. Enzymes are important in many industrial applications, so it becomes essential to discover novel ways of creating more robust versions that will perform their chemical reactions. The AUC will help us identify enzymes that have been re-engineered to be more stable. We will use the AUC to study how specialised human proteins called TIP48 and TIP49 associate with each other and how this is modified by small molecules. Both proteins use chemical energy to perform their role in large nuclear complexes. Oligomerisation is crucial to couple ATP hydrolysis to the molecular action of these proteins. A heat-stable form of TIP49 in archaeal organisms will be studied to discover both the importance of these small molecules for association processes and also the effect of deleting part of TIP49 on its subunit organisation. A different set of proteins are involved in mitosis, the process of cell division. The AUC will be invaluable for defining how these mitotic complexes are formed and their stability, and this work is crucial to lead to more detailed molecular structures that will be determined by other methods.