Abstract
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 non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, 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. We disentangle specific and non-specific effects that control Hsp90's ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins.
Highlights
Protein function is essential for life as we know it
All three modulations provoke a similar steady-state behavior, namely an increase in Hsp90’s closed conformation and in Hsp90’s ATPase rate. They show significant differences in their kinetics, which could be revealed by single-molecule FRET
This can be rationalized by the emerging picture of yeast Hsp90, as a very flexible dimer that relies critically on external assistance to control its non-productive flexibility
Summary
Protein function is essential for life as we know it. It is largely encoded in a protein’s amino-acid chain that dictates the specific 3D structure, and the conformational flexibility and dynamics of a protein in a given environment. After translation by the ribosome, protein function depends strongly on post-translational modifications (PTMs) (Deribe et al, 2010), and on binding of nucleotides (Hodge and Ridley, 2016), cofactors (Weikum et al, 2017), various protein-protein interactions (PPIs) (Scott et al, 2016), and global effects, such as temperature (Danielsson et al, 2015), macro-molecular crowding and phase separation (Uversky, 2017), redox conditions (Levin et al, 2017), osmolarity (de Nadal and Posas, 2015) etc This regulation occurs on very diverse levels of localization. We take a step and disentangle how a PTM-related point mutation, a co-chaperone interaction, and macro-molecular crowding affect the function, kinetics, and thermodynamics of this multi-domain protein
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