Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Nov 23;96(24):13732-7.
doi: 10.1073/pnas.96.24.13732.

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network

P Goloubinoff  1 , A Mogk, A P Zvi, T Tomoyasu, B Bukau
Affiliations

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network

P Goloubinoff et al. Proc Natl Acad Sci U S A. .

Abstract

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Aggregation and disaggregation of MDH. (A) Time-dependent inactivation and aggregation of 720 nM MDH at 47°C in the absence of chaperones. (B) Time-dependent disaggregation at 25°C of heat-aggregated MDH from A without or in the presence of supplemented 83 nM ClpB6, 1 μM DnaK, 0.2 μM DnaJ, 0.1 μM GrpE, and 2 mM ATP. (NH4)2SO4 (100 mM) was added or not, at t = 10 min. Turbidity measured at t = 1 min was set as 100%. (C) Effect of order-of-addition of ClpB6 and KJE on the rate of MDH disaggregation (as in B). Given yields correspond to regained MDH activity after 3-hr incubation at 25°C. (D) Time-dependent reactivation of MDH (expressed in percentage of initial native 720 nM MDH) in the presence of ATP, ClpB and/or KJE, as in B. (E) Effect of increasing concentrations of ClpB6 in presence of constant amounts of KJE and MDH on the rate of MDH reactivation (as in D). (F) Effect of increasing concentrations of MDH aggregates in the presence of constant amounts of ClpB6 and KJE on the rate of MDH reactivation (as in D).
Figure 2
Figure 2
Inhibition of MDH disaggregation by sulfate and centrifugation. (ClpB+KJE)-mediated MDH reactivation as in Fig. 1 was measured after 270 min. Addition of 100 mM (NH4)2SO4 or centrifugation (3 min, 16,000 × g) was performed at indicated time points. Inhibition of reactivation was expressed as percent loss of recovered MDH activity as compared with the activity without inhibition at 270 min.
Figure 3
Figure 3
Modulation of interaction and ClpB-induced conformational changes in heat-aggregated MDH. Time-dependent change in bis-ANS binding (A) and Congo red binding (B) to heat-aggregated MDH after incubation with ATP, ClpB and/or KJE as in Fig. 1B. bis-ANS and Congo red binding to aggregated MDH before chaperone addition is set as 100%; baseline binding levels in native MDH is set as 0%. (C) Interaction of ClpB6 with MDH aggregates and nucleotides by Trp fluorescence. Effects of increasing concentrations of aggregated MDH on the intrinsic fluorescence intensity of ClpB6 (83 nM) without or with 3 mM ATP or ADP.
Figure 4
Figure 4
Solubilization of a wide range of heat-aggregated proteins. Soluble 35S-labeled protein extracts from wild-type E. coli cells (MC4100) were aggregated at 45°C and incubated for 4 hr with ATP, ClpB, and KJE (see Experimental Procedures). Resolubilized and aggregated proteins were separated by centrifugation. Gels were loaded with equal volumes, such that the insoluble fractions were 2.5-fold more concentrated relative to the soluble fractions. Soluble (S) and aggregated (P) proteins were analyzed by Coomassie staining (Upper) and quantified by scintillation counting (Lower).
Figure 5
Figure 5
Model for the sequential action of ClpB and KJE. ClpB binds protein aggregates. ATP-driven changes in the structure of ClpB expose new hydrophobic sites in ClpB-bound aggregates. This allows binding of KJE and mediating of disaggregation and refolding of the aggregated protein.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bukau B, Horwich A L. Cell. 1998;92:351–366. - PubMed
    1. Jaenicke R. Biol Chem. 1998;379:237–243. - PubMed
    1. Turnell W G, Finch J T. J Mol Biol. 1992;227:1205–1223. - PubMed
    1. Horwich A L, Weissman J S. Cell. 1997;89:495–510. - PubMed
    1. Woo K M, Kim K I, Goldberg A L, Ha D B, Chung C H. J Biol Chem. 1992;267:20429–20434. - PubMed

Publication types

LinkOut - more resources