Preparing chaperone—client protein complexes for biophysical and structural studies

Many proteins are prone to misfolding or unfolding and aggregation in the aqueous environment of the cell, either due to their complex ternary structure, cellular localization, molecular crowding, cellular stress, or their intrinsic hydrophobic nature. Molecular chaperones are involved in biogenesis of such proteins, and in ensuring their intactness throughout their life cycle. While there are many different types of chaperones with various cellular roles, essentially all of them are characterized by a basic “holdase” function, i.e. the ability to bind to proteins that are generally in some non-native conformation – often these are called “client” proteins. Understanding how these complexes form, what (residual) structure the client proteins have when bound, and how the client protein may eventually be released again from the chaperone is essential for resolving questions in biogenesis of cells and organelles. The preparation of chaperone complexes for in vitro studies is essential for characterising their function at the structural level. However, preparing these complexes is often a significant challenge in itself. Here, we propose a systematic view of the factors that can counteract chaperone--client complex formation, and identify possible routes for generating such complexes. We review the various methods that have been used in previous studies. This review will be of interest for anyone trying to obtain chaperone complexes for structural or biochemical studies.