Same Equilibrium. Different Kinetics. Protein Functional Consequences

Abstract

12 In a living cell, protein function is regulated in several ways, including post-translational 13 modifications (PTMs), protein-protein interaction, or by the global environment, e.g. by 14 crowding or phase separation. While site-specific PTMs act very locally on the protein, spe-15 cific protein interactions typically affect larger (sub-)domains, and global changes affect the 16 whole protein non-specifically. 17 Herein, we directly observe protein regulation with three different degrees of localization, 18 and present the effect on the Hsp90 chaperone system on the level of conformational equi-19 libria, kinetics and protein function. Interestingly, we find by single-molecule FRET that sim-20 ilar functional and conformational steady-state observations are caused by completely dif-21 ferent underlying kinetics. Solving the complete kinetic rate model allows us to disentangle 22 specific and non-specific effects controlling Hsp90's ATPase function, which has remained 23 puzzling up to date. Lastly, we introduce a new mechanistic concept: functional stimulation 24 through conformational confinement. Our results highlight how cellular protein regulation 25 works by fine-tuning the conformational state space of proteins. 26Protein function is essential for life as we know it. It is largely encoded in a protein's amino-acid 27 chain that dictates not only the specific 3D structure, but also the conformational flexibility and dy-28 namics of a protein in a given environment. Precise regulation of protein function is vital for every 29 living cell to cope with an ever-changing environment, and occurs on many levels pre-and post-30 translationally 1,2 . After translation by the ribosome, protein function depends strongly on post-31 translational modifications (PTMs) 3 , but also on binding of nucleotides 4 , cofactors 5 , various protein-32 protein interactions (PPIs) 6 , and global effects, such as temperature 7 , macro-molecular crowding 33 and phase separation 8 , redox conditions 9 , osmolarity 10 etc. Importantly, this regulation occurs on 34 very diverse levels of localization. Global effects affect the whole protein non-specifically, PPIs act 35 at a given interface and site-specific modifications are very localized. Nevertheless, all of them in-36 fluence the molecular properties that determine the 3D conformation, the conformational dynamics, 37 and thereby also the function of a protein [11][12][13][14][15] . The chaperone protein Hsp90 16 is an excellent test 38 system to investigate diverse regulation mechanisms 17 . It was recently discussed that a single 39 PTM can functionally mimic a specific co-chaperone interaction in human Hsp90 18 . Here we take a 40 next step and disentangle how a PTM-related point mutation, a co-chaperone interaction, and 41 macro-molecular crowding affect the function, kinetics, and thermodynamics of this multi-domain 42 protein. 44Hsp90 is an important metabolic hub. Assisted by about twenty known cochaperones, yeast Hsp90 45 is involved in the maturation of 20% of the entire...