Kinetics of Molecular Chaperone Action

Schmid, Daniel; Baici, Antonio; Gehring, Heinz; Christen, Philipp

doi:10.1126/science.8310296

Cited by 453 publications

(477 citation statements)

References 15 publications

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“…4 B-D), the extended conformation of rhodanese disappears with a rate constant of (2.5 ± 0.4) 10 −3 s −1 (Table S1). This rate is in a similar range as previously determined off-rates for substrates from ADP-bound DnaK (38,47,48). After reaching equilibrium, the transfer efficiency histograms are indistinguishable from those of the DnaJ-rhodanese complex, suggesting complete dissociation of DnaK (compare Fig.…”

Section: Dynamics Of Complex Formation From Microfluidicsupporting

confidence: 86%

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Kellner

Hofmann

Barducci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

118

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Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ-DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Hsp70 | Hsp40 | Förster resonance energy transfer | protein folding | FRET

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Section: Dynamics Of Complex Formation From Microfluidicsupporting

confidence: 86%

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Kellner

Hofmann

Barducci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

118

View full text Add to dashboard Cite

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“…Earlier studies showed that DnaJ deletion mutants that lack the complete zinc binding region are still capable of interacting with substrate proteins such as 32 (18). This suggested that the low affinity binding site might be located in the C terminus of DnaJ.…”

Section: Discussionmentioning

confidence: 99%

“…5B). This is due to the negligible affinity of DnaK-ATP complexes to substrate proteins (32,33). The presence of both DnaJ and DnaK in a 0.5:10:1 molar ratio of DnaJ:DnaK:luciferase, however, completely suppressed luciferase aggregation (compare Fig.…”

Section: Fig 4 the Dnaj Zinc Center II Is Important For The Functiomentioning

confidence: 99%

The Roles of the Two Zinc Binding Sites in DnaJ

Linke

Wolfram

Bussemer

et al. 2003

Journal of Biological Chemistry

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All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/ GrpE foldase machinery catalytically active.

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“…1A) (Schmid et al 1994;Gisler et al 1998;Mayer et al 2000b). Spontaneous transition between the two states is extremely slow but is stimulated synergistically by substrates and DnaJ (Karzai and McMacken 1996;Laufen et al 1999).…”

Section: Introductionmentioning

confidence: 99%

The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Tomita

2007

Syst Synth Biol

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Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures.

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Kinetics of Molecular Chaperone Action

Cited by 453 publications

References 15 publications

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

The Roles of the Two Zinc Binding Sites in DnaJ

The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

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