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. 2012 Jul;21(7):1042-55.
doi: 10.1002/pro.2087. Epub 2012 Jun 11.

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Ashok Sekhar  1 , Margarita Santiago, Hon Nam Lam, Jung Ho Lee, Silvia Cavagnero
Affiliations

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Ashok Sekhar et al. Protein Sci. 2012 Jul.

Abstract

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H(D). Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H(D). We find that K/J/E slows down RNase H(D)'s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H(D) with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.

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Figures

Figure 1
Figure 1
A: Three-dimensional structure of E.coli RNase H (PDB code: 1F21). The N and C termini are labeled. The unique DnaK binding site is shown in green. B: Predicted local DnaK binding scores of RNase HD. Regions with a score lower than −5 are potential strong DnaK binding sites. The secondary structure of wild type E.coli RNase H derived from the protein crystal structure is mapped above the graph.
Figure 2
Figure 2
Experimental far-UV CD-detected stopped-flow kinetic traces for the refolding of RNase HD (blue) in the absence and presence of various combinations of DnaK, DnaJ, and GrpE at 30°C at 222 nm. Time-dependent variations in ellipticity of refolding buffer and urea-unfolded RNase HD are shown in red and orange, respectively. The ellipticity of the refolding buffer subtracted from the ellipticity of the refolding trace, denoted as Δellipticity, is plotted on the y-axis. Hence, the ellipticity of the refolding solution, containing contributions from chaperones, has been subtracted out. The observed time-dependent change in ellipticity thus arises from the substrate protein alone, assuming that the secondary structure of the chaperones is not significantly perturbed. Final concentrations of RNase HD, DnaK, DnaJ, and GrpE and ATP in the refolding mixture were 5, 15, 3, and 6 μM and 1 mM, respectively. Least squares fits of the kinetic traces to linear or single-exponential functions are in black. Average rate constants derived from curve fitting are listed close to each corresponding refolding trace. Error bars represent 1 standard error calculated from 2 to 8 independent repeats, with each repeat comprising 4–6 replicate measurements.
Figure 3
Figure 3
Stopped-flow refolding data analysis. Bar diagram reporting the (A) rate constants, (B) final (ηF) ellipticity fractions [see Eqs. (1) and (2)], and (C) burst phase amplitudes (ηB), and obtained from stopped-flow data analysis. Chaperone combinations pertinent to each experimental condition are listed below the corresponding bar. Error bars represent 1 standard error calculated from 2 to 8 independent measurements, with each measurement consisting of 4–6 independent runs. Statistically significant differences (relative to the RNase HD-only experiment) are shown in the graph by asterisks (*P ≤ 0.1, **P ≤ 0.05, ***P ≤ 0.001).
Figure 4
Figure 4
Analytical gel filtration chromatograms, followed by electronic absorption at 214 nm, of RNase HD (A) in the absence of chaperones, (B) in the presence of DnaK/ATP, and (C) in the presence of DnaK, DnaJ, and ATP. Urea-unfolded RNase HD was refolded in 20 mM sodium acetate pH 6.0 containing 100 mM KCl, 5 mM MgCl2, and 1 mM ATP in the absence (A) and presence (B and C) of chaperones before injecting into the size-exclusion column. Peaks denoted with asterisks arise from buffer components (i.e., ATP and ADP). The elution volume corresponding to each molecular-weight standard used to calibrate the column is indicated at the top of each panel (see Materials and Methods for names of standard proteins). In all chromatograms, absorption intensities are reported on the same absolute scale.
Figure 5
Figure 5
Analytical reverse-phase HPLC chromatograms of gel filtration fractions A and B [see Fig. 4(A,C)].
Figure 6
Figure 6
SDS-PAGE gel analysis of gel filtration fractions A and B [see Fig. 4(A,C)]. Prior to running each of the gel filtration fractions through the gel (16.5% Tris-tricine), precipitation with trichloroacetic acid (TCA) was carried out to effectively concentrate the samples for SDS-PAGE analysis. The arrows indicate bands corresponding to RNase HD.
Figure 7
Figure 7
Extent of complex formation between RNase HD and the DnaK chaperone measured by gel filtration as the reduction in native RNase HD peak intensity. Error bars correspond to ±1 standard error, derived from 2 to 7 independent measurements.
Figure 8
Figure 8
A: Enzymatic activity of RNase HD measured after 10 min from refolding initiation, in the presence of different combinations of chaperones. Data are shown as percent of the activity of a control RNase HD sample refolded in the absence of chaperones. Error bars represent ± 1 standard error of the mean, calculated from three independent measurements. B: Computer simulations predicting the expected ATP depletion as a function of time under the stopped-flow and gel filtration/activity assay experimental conditions. The refolding time in the stopped-flow experiments is 160 s, while the refolding time in the gel filtration and activity assays is 10 min. The spatial separation of the solution components during gel filtration experiments may introduce additional effects (not taken into account in this simulation).
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