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. 2012 Jun 5;109(23):8937-42.
doi: 10.1073/pnas.1201380109. Epub 2012 May 21.

Global analysis of chaperone effects using a reconstituted cell-free translation system

Tatsuya Niwa  1 , Takashi Kanamori, Takuya Ueda, Hideki Taguchi
Affiliations

Global analysis of chaperone effects using a reconstituted cell-free translation system

Tatsuya Niwa et al. Proc Natl Acad Sci U S A. .

Abstract

Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. A dataset that evaluates which chaperones are effective for aggregation-prone proteins would provide an invaluable resource not only for understanding the roles of chaperones, but also for broader applications in protein science and engineering. Therefore, we comprehensively evaluated the effects of the major Escherichia coli chaperones, trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ∼800 aggregation-prone cytosolic E. coli proteins, using a reconstituted chaperone-free translation system. Statistical analyses revealed the robustness and the intriguing properties of chaperones. The DnaK and GroEL systems drastically increased the solubilities of hundreds of proteins with weak biases, whereas trigger factor had only a marginal effect on solubility. The combined addition of the chaperones was effective for a subset of proteins that were not rescued by any single chaperone system, supporting the synergistic effect of these chaperones. The resource, which is accessible via a public database, can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
An in vitro expressed proteome approach for global aggregation analysis. Schematic illustration of the experiment. Seven hundred and ninety-two aggregation-prone proteins were separately expressed with a reconstituted cell-free translation system, the PURE system, in the absence and the presence of the major E. coli chaperones (trigger factor, TF; DnaK/DnaJ/GrpE, KJE, GroEL/GroES, GroE). Each translation product was labeled with [35S]methionine. After translation, the uncentrifuged total fraction (Total) and the supernatant fraction after centrifugation (Sup) were electrophoresed and quantified by autoradiography. The ratio of the translation products in the Total and Sup fractions was defined as the solubility, which represented the aggregation propensity of the protein. The dataset (∼800 × 4) obtained from this experiment was subjected to statistical analyses to investigate the relationship between the effects of chaperones and the various properties of the proteins.
Fig. 2.
Fig. 2.
Global analysis of chaperone effects on the prevention of aggregate formation. (A) Typical examples. SDS-gels of four aggregation-prone E. coli cytosolic proteins (asd, hemB, yedS, and yajB) in the absence and the presence of the chaperones are shown. The numbers below the electrophoretic pattern indicate the solubility values, calculated by the ratio of the amount of translation products in the Sup (S) and Total (T) fractions. (B) Histograms of solubilities in the presence of three E. coli chaperones. The aggregation-prevention effect is represented as Δsolubility, defined by subtracting the solubilities in the absence of chaperones from those in the presence of each chaperone (see Fig. S2B for raw data).
Fig. 3.
Fig. 3.
Overlaps and differences in chaperone effects. (A), A Venn diagram showing the overlap in the effects of chaperones. The numbers of proteins with solubilities that were drastically increased by at least one chaperone (defined as > +50% Δsolubility) are shown. See also Table S1. (B) Two-dimensional distribution plot of Δsolubilities for DnaKJE and GroE. Dashed lines represent the boundaries of the lower and upper quartiles [34, 67% and 26, 58% solubility values for DnaKJE (green) and GroE (purple), respectively].
Fig. 4.
Fig. 4.
Correlation between chaperone effects and physicochemical properties. (A) Histograms of molecular weight for all evaluated proteins and the proteins that were rescued by DnaKJE or GroE. Well-solubilized proteins were defined as those in the upper quartile (≥75th percentile) in Δsolubility for DnaKJE or GroE. (B) Comparison between Δsolubility and SCOP classes (all α, all β, α/β, and α+β). The distributions of Δsolubility for DnaKJE and GroE are shown by Kernel-type density maps. The numbers in parentheses indicate the number of proteins categorized in each class. (C) Comparison between Δsolubility and SCOP folds. The four most abundant SCOP folds in the quantified proteins are shown by Kernel-type density maps. The numbers in parentheses indicate the number of proteins categorized in each fold. a4, DNA/RNA-binding 3-helical bundle; c1, TIM β/α-barrel; c37, P-loop containing nucleoside triphosphate hydrolases; c94, Periplasmic binding protein-like II.
Fig. 5.
Fig. 5.
Combined effects of chaperones on recalcitrant proteins. Fifty-three proteins, with Δsolubility in the presence of DnaKJE or GroEL that was lower than the boundaries of the lower quartiles of both DnaKJE and GroE (lower left area in Fig. 3B), were chosen as recalcitrant proteins. (A) Typical examples of the combination effect of chaperones on several recalcitrant proteins (nhsE, ybbB, and mhpR). The numbers below the electrophoretic pattern indicate the solubility values. T&G, TF and GroEL/ES; T&K, TF and DnaKJE; G&K, GroEL/ES, and DnaKJE; T&G&K, TF, GroEL/ES, and DnaKJE. (B) Histograms of Δsolubility obtained from the combination of chaperones.
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