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

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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.