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. 2012 Oct;21(10):1489-502.
doi: 10.1002/pro.2139.

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

Ashok Sekhar  1 , Hon Nam Lam, Silvia Cavagnero
Affiliations

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

Ashok Sekhar et al. Protein Sci. 2012 Oct.

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.

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Figures

Figure 1
Figure 1
Cartoon representation of the kinetic partitioning hypothesis underlying the computational model of protein folding mediated by the K/J/E chaperone system. The unfolded substrate partitions between competing folding and chaperone binding pathways en route to the native state.
Figure 2
Figure 2
Three-dimensional structures and DnaK-binding scores of (A) RNase HD (PDB ID: 1F21), (B) drkN SH3 (PDB ID: 2A36), and (C) ubiquitin (PDB ID: 1UBQ). The predicted local DnaK binding site of each protein is shown in red.
Figure 3
Figure 3
Experimental assessment of client protein–DnaK interaction (A) during refolding and (B) at equilibrium for the three substrates: RNase HD, drkN SH3, and ubiquitin. The interaction between the client protein and the DnaK chaperone was quantitatively evaluated as the percent reduction in the native protein SEC peak intensity (See the Materials and Methods section). For each reversible step, arrow lengths denote the relative rates of the forward and backward steps.
Figure 4
Figure 4
(A) Schematic representation of the interactions between DnaK, DnaJ, GrpE, and substrate present in the CHAMP70 computational model. Numbers have been assigned to reactions, and the corresponding rate constants are listed in Table I. The abbreviation for each molecular species is noted in red. (B) Computationally predicted time-course for the population of various chaperone–substrate complexes during the folding of the representative substrate protein mAcP in the presence of the K/J/E chaperone system. The molecular species corresponding to each trace of the plot is color-coded as shown. Protein folding is initiated at time t = 0.
Figure 5
Figure 5
Comparison between experimentally observed and computationally predicted (A) stopped-flow rate constants and (B) SEC-detected transient complex formation associated with the interaction of RNase HD with K/J/E. Experimental data are reproduced from Sekhar et al. Computational predictions were generated via CHAMP70. Black bars denote experimental data and hatched bars are CHAMP70 predictions, derived without using any adjustable parameters. The gray bars in (A) are computational predictions derived by fitting CHAMP70 to the corresponding experimental data, using kon and koff as adjustable parameters.
Figure 6
Figure 6
Plot illustrating the known folding rates (ln kf) and thermodynamic stabilities (ΔG0UN) of 59 two-state-folding proteins studied computationally (black). The dashed vertical and horizontal lines denote the boundaries between the four different regimes (fast-folding/stable, slow-folding/stable, slow-folding/unstable, and fast-folding/unstable) identified by CHAMP70, for the interaction of two-state-folding substrate proteins with the K/J/E chaperone system. The three proteins studied experimentally are also shown (color-coded according to Fig. 2).
Figure 7
Figure 7
Plot illustrating the computationally predicted maximum percent of chaperone-associated substrate during folding (A and B) and at equilibrium (C and D), as functions of the substrate folding rate constant (ln kf, A and C) and thermodynamic stability (ΔG0UN, B and D).
Figure 8
Figure 8
Three-dimensional histograms representing the computationally predicted percent of chaperone-associated substrate as a function of the binding rate constant (kon) and dissociation constant (Kd) for the substrate's interaction with ATP-DnaK, in the case of (A) slow-folding and (B) fast-folding substrates. The amount of chaperone-associated client protein was measured after time t = 1/kf, following folding initiation.
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