Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

Abstract

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli's Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H(D) as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k(f) <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰ (UN) ≥-2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.