Proteomics

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DnaK interaction and folding of firefly luciferase studied by hydrogen/deuterium exchange – mass spectrometry

ABSTRACT: The ATP-dependent chaperones of the Hsp70 class (DnaK in E. coli) function in protein folding in cooperation with J proteins and nucleotide exchange factors (DnaJ and GrpE in E. coli, respectively). Hsp70 prevents protein aggregation, increasing the folding yield, but whether it also enhances the rate of folding is unclear. Equilibrium hydrogen/deuterium exchange – mass spectrometry showed that DnaK stabilizes a poorly structured state of firefly luciferase (FLuc). Pulsed-label experiments, together with orthogonal analyses by spFRET, identified compact inter-domain misfolded states of FLuc that convert slowly to the native state. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. By resolving misfolding and accelerating folding the Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: David Balchin  

LAB HEAD: F. Ulrich Hartl

PROVIDER: PXD016509 | Pride | 2020-01-15

REPOSITORIES: Pride

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Dataset's files

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Action DRS
20190121_S2_DB_01036_LucNative_Undeut_A.raw.zip Raw
20190121_S2_DB_01037_LucNative_10sec_A.raw.zip Raw
20190121_S2_DB_01038_LucNative_30sec_A.raw.zip Raw
20190121_S2_DB_01039_LucNative_100sec_A.raw.zip Raw
20190121_S2_DB_01040_LucNative_300sec_A.raw.zip Raw
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Publications

Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein.

Imamoglu Rahmi R   Balchin David D   Hayer-Hartl Manajit M   Hartl F Ulrich FU  

Nature communications 20200117 1

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) ~20-fold over the rate of spontaneous folding measured in the absence of  ...[more]

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