The Small Heat-shock Protein IbpB from Escherichia coli Stabilizes Stress-denatured Proteins for Subsequent Refolding by a Multichaperone Network

Sophia Diamant,Johannes Buchner,Pierre Goloubinoff,Lea Veinger

doi:10.1074/jbc.273.18.11032

Sophia Diamant, Johannes Buchner + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.18.11032

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Abstract

The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.

Highlights

The small heat-shock proteins1 belong to a ubiquitous family of low molecular mass (15–30 kDa), stress-induced proteins in prokaryotes and eukaryotes
IbpB-stabilized malate dehydrogenase (MDH) Can be Refolded by KJE ϩ ATP—When exposed to 47 °C, the thermolabile enzyme MDH is inactivated at an apparent rate of 0.13 minϪ1 [11] and forms large insoluble protein aggregates that do not refold spontaneously after the heat shock
We found that the IbpB1⁄7MDH did not significantly reactivate, even after a 5-h incubation at room temperature (Fig. 2A)

Summary

EXPERIMENTAL PROCEDURES

Phage Complementation—Wild type and E. coli B mutant T850 [22] were used for in vivo complementation assays of T4 morphogenesis as described in Ref. 23. Chaperone Activity Assays—MDH was heat-denatured for 30 min at 47 °C as in Ref. 11 in the presence of various amounts of IbpB, DnaK, DnaJ, and/or GroEL, GroES as specified and subsequently refolded at 25 °C in the presence of supplemented DnaK/DnaJ/GrpE and/or GroEL/ GroES (4, 4, 1, 4, and 6 ␮M, respectively) in 50 mM triethanolamine, pH 7.5, 20 mM MgAc2, 150 mM KCl, 5 mM dithiothreitol, 3 mM phosphoenol pyruvate, 7 ␮g/ml pyruvate kinase (Sigma), and 2 mM ATP (buffered with KOH), as indicated. Enzymatic Assays—The activity of MDH was measured as described in Ref. 27, at 25 °C in 150 mM potassium phosphate buffer, pH 7.5, 10 mM dithiothreitol, 0.5 mM oxaloacetate, and 0.28 mM NADH (Sigma). A Superose 6B gel filtration column (Pharmacia) (at 0.5 ml minϪ1), run in the presence of the refolding buffer and 1 mM ATP was used for separation. 3H-Labeled samples were collected at the indicated elution volumes (Fig. 5), mixed with a 6-fold volume of LumaxTM, and counted

RESULTS

TABLE I Apparent rates of MDH refolding after heat denaturation

ATP ϩ IbpB ϩ ELS No

IbpB IbpB IbpB L KJ KJ

DISCUSSION