Mechanisms of chaperone assisted protein folding and unfolding studied by electron cryo microscopy and native ion mobility mass spectrometry

该项目的目的是通过伴侣和底物多肽之间的分子相互作用，了解辅助蛋白质展开和折叠的机制。具体地说，关键的伴侣是如何通过与序列和折叠基本无关的机制，利用ATP结合和水解的能量来解聚、展开或折叠其他蛋白质的？随着电子冷冻显微镜和大组装的质谱学领域的重大进展，结构生物学和动力学已经进入了一个新的水平，分离的大分子复合体和细胞环境中的分子结构的分辨率大大提高。我们希望将这些最先进的方法应用于质量控制和折叠中难以捉摸的、瞬时的复合体。低温电子显微镜能够在体外和原位对不同种类的复合体进行分类和分类。由于辅助折叠中的中间络合物必然含有非天然和无序的组分，这一具有挑战性的结构项目需要分离多个状态。使用MS One可以识别蛋白质复合体的化学计量比，确定这些复合体的拓扑结构，并在增加离子迁移率的情况下，探测构象变化和监测蛋白质展开事件。离子迁移率和质谱学相结合的方法是检测我们在这里建议研究的多相、非共价组装的理想方法。然而，用于处理数据的软件仍处于初级阶段，需要进一步开发以提取此类数据集中存在的丰富信息，这是我们在这个项目中计划做的。合适的复合体可以由细菌伴侣蛋白GroEL和GroES与非天然底物形成，也可以产生在错误折叠疾病(如帕金森氏症和其他神经退行性疾病，通常涉及淀粉样纤维的形成)所涉及的聚集体上操作的展开酶复合体。这些复合体的聚集和解聚是基本的和主要的生物医学问题，其分子基础仍然知之甚少。

The aim of the project is to understand the machinery of assisted protein unfolding and folding, through the molecular interactions between chaperones and substrate polypeptide. Specifically, how do key chaperones use the energy of ATP binding and hydrolysis to disaggregate, unfold or fold other proteins, via mechanisms that are largely independent of sequence and fold?With the major recent advances in field of electron cryomicroscopy as well as in mass spectrometry of large assemblies, structural biology and dynamics have moved to a new level, with greatly improved resolution of isolated macromolecular complexes and of molecular machinery in the cellular environment. We wish to apply these state of the art methods to the elusive, transient complexes in quality control and folding. CryoEM enables sorting and classification of heterogeneous complexes, both in vitro and in situ. Since the intermediate complexes in assisted folding necessarily contain non native and disordered components, this challenging structural project requires separation of multiple states. Using MS one can identify the stoichiometry of protein complexes, determine the topology of these complexes and, with the addition of ion mobility, probe conformational changes and monitor protein unfolding events. The combined ion mobility and mass spectrometry approach is ideal for examining heterogeneous, non covalent assemblies that we propose to study here. However, the software used to process the data is still in its infancy and requires further development to extract the wealth of information present in such datasets, something we plan to do in this project.Suitable complexes can be produced of bacterial chaperonins GroEL and GroES with non native substrates, and also complexes of unfoldases operating on the aggregates involved in misfolding diseases such as Parkinson's and other neurodegenerative conditions, typically involving the formation of amyloid fibres. Accumulation and disaggregation of these complexes are fundamental and major biomedical problems for which the molecular basis is still very poorly understood.