Preparing Chaperone–Client Protein Complexes for Biophysical and Structural Studies

Understanding the mechanisms of chaperones at the atomic level generally requires producing chaperone–client complexes in vitro. This task comes with significant challenges, because one needs to find conditions in which the client protein is presented to the chaperone in a state that binds and at the same time avoid the pitfalls of protein aggregation that are often inherent to such states. The strategy differs significantly for different client proteins and chaperones, but there are common underlying principles. Here, we discuss these principles and deduce the strategies that can be successfully applied for different chaperone–client complexes. We review successful biochemical strategies applied to making the client protein “binding competent” and illustrate the different strategies with examples of recent biophysical and biochemical studies.