Same Equilibrium. Different Kinetics. Protein Functional Consequences.

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein in non-specific ways. Herein, we directly observe protein regulation in three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational equilibria, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady-states are caused by completely different underlying kinetics. Solving the complete kinetic rate model allows us to disentangle specific and non-specific effects controlling Hsp90s ATPase function, which has remained a puzzle up to this day. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results highlight how cellular protein regulation works by fine-tuning the conformational state space of proteins. SignificanceProteins are perceived more and more as dynamic systems whose function depends critically on local and global flexibility. While 3D structures of proteins are frequently available today, our models often lack the time component, namely rate constants that determine protein function and regulation. Here we used single-molecule FRET to elucidate how the chaperone protein Hsp90 is regulated on various levels, locally and globally. We find that ATPase stimulation occurs not only through specific interactions, but also non-specifically by reducing non-productive conformational flexibility; i.e. by changing kinetics rather than thermodynamics. Our work introduces stimulation through conformational confinement as a general mechanistic concept. We anticipate that this concept plays an important role in protein regulation, phase separation, and in dynamic protein systems in general.