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

Kellner, Ruth; Hofmann, Hagen; Barducci, Alessandro; Wunderlich, Bengt; Nettels, Daniel; Schuler, Benjamin

doi:10.1073/pnas.1407086111

Cited by 111 publications

(147 citation statements)

References 61 publications

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“…S4 C and D) in which hTRF1 first unfolds and then binds one molecule of DnaK with an affinity of 1.4 ± 0.2 μM. The globally unfolded conformation of DnaK-bound hTRF1 observed in our experiments is consistent with previous low-resolution circular dichroism (13,14) and singlemolecule fluorescence data (15) showing that DnaK-bound clients are expanded and have a diminished helical content compared with the native substrate. In addition, the 1:1 stoichiometry of the DnaKhTRF1 complex is in line with expectations from the small size of hTRF1 and the presence of only one strong DnaK binding site along its primary sequence (Fig.…”

Section: Resultssupporting

confidence: 90%

“…2A) confirm that this substrate exists in a globally unfolded conformation when bound to DnaK. However, on average, 15 N, 1 HN, and 13 C β chemical shifts from DnaK-bound hTRF1 are smaller than random coil values whereas the corresponding 13 C α and 13 C′ shifts are larger, consistent with residual helical structure in the DnaK-bound conformation. To estimate the secondary structure content in the DnaK-bound hTRF1 ensemble, we computed the secondary structural propensity (SSP) score (21) using chemical shifts of the bound state.…”

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 83%

“…Upon addition of ATP-DnaK T199A to a sample of 15 N hTRF1 the size of the minor dips significantly increased, clearly demonstrating the presence of the ATP-DnaK T199A -bound form (Fig. 4B and Fig.…”

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 88%

“…The main dip is located at the position of the chemical shift of the resonance belonging to the major state conformer and a smaller dip at the position of the frequency of the corresponding excited state. 15 N, 13 C α , and 13 C′ CEST profiles of hTRF1 were acquired in the absence of DnaK at 35°C (Fig. 3A and Fig.…”

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 99%

“…Larger fragments of apomyoglobin bound to the DnaK SBD were found to be predominantly unfolded but adopted small amounts of native and nonnative helical structure (11,12). In addition, low-resolution studies on protein substrates have suggested that the substrate is globally unfolded (13,14) and expanded in the DnaK-bound conformation (15). Although these studies have provided important insights into DnaK-substrate binding, a more comprehensive structural characterization of this interaction is crucial for a detailed understanding of DnaK function.…”

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confidence: 99%

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Mapping the conformation of a client protein through the Hsp70 functional cycle

Sekhar

Rosenzweig

Bouvignies

et al. 2015

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

109

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The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP-and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaKbound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.Hsp70 | protein folding | NMR | molecular chaperones | CEST T he Hsp70 chaperone system forms a central hub in the cellular proteostasis network, performing a large number of functions, all of which are predicated upon its relatively simple ATPdependent interaction with a client protein (1, 2). Hsp70 is a 70-kDa protein containing both nucleotide (NBD) and substrate (SBD) binding domains connected by a conserved linker (2). The beststudied Hsp70 is Escherichia coli DnaK, which recognizes an approximate seven-residue protein segment rich in large aliphatic hydrophobic residues flanked by positively charged amino acids (3). Client substrates enter the Hsp70 functional cycle by binding the ATP form of the chaperone, which has lower substrate affinity but faster binding and release rates compared with the ADP state (2). The crystal structure of ATP-DnaK (4, 5) shows that the two domains of Hsp70 are docked on each other, with the helical lid flanking the DnaK binding cleft in an open state. Subsequent ATP hydrolysis, stimulated by both Hsp40 and the bound substrate, locks the substrate in the binding pocket (2) and disengages the two domains of DnaK from each other, leading to major structural differences between the ATP-(4, 5) and ADP-bound (6) forms of the chaperone. Finally, ADP release, facilitated by nucleotide exchange factors (7), and rebinding of ATP free the bound substrate and reset the chaperone cycle.Although the structural transitions of DnaK and the role of allostery throughout the chaperone reaction cycle are becoming increasingly well understood (7), far less information is available on the substrate conformation in the DnaK-bound state. Our current view is derived primarily from studies on the isolated SBD of DnaK in conjunctio...

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Section: Resultssupporting

confidence: 90%

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 83%

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 88%

Section: Dnak-bound Htrf1 Contains Substantial Residual Secondary Strmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Mapping the conformation of a client protein through the Hsp70 functional cycle

Sekhar

Rosenzweig

Bouvignies

et al. 2015

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

109

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Recent advances in understanding catalysis of protein folding by molecular chaperones

2020

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Molecular chaperones are highly conserved proteins that promote proper folding of other proteins in vivo. Diverse chaperone systems assist de novo protein folding and trafficking, the assembly of oligomeric complexes, and recovery from stress‐induced unfolding. A fundamental function of molecular chaperones is to inhibit unproductive protein interactions by recognizing and protecting hydrophobic surfaces that are exposed during folding or following proteotoxic stress. Beyond this basic principle, it is now clear that chaperones can also actively and specifically accelerate folding reactions in an ATP‐dependent manner. We focus on the bacterial Hsp70 and chaperonin systems as paradigms, and review recent work that has advanced our understanding of how these chaperones act as catalysts of protein folding.

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Rapid Microfluidic Dilution for Single‐Molecule Spectroscopy of Low‐Affinity Biomolecular Complexes

et al. 2017

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To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed amicrofluidic device that allows aconcentrated sample to be diluted by up to five orders of magnitude within milliseconds,atthe physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and threecolor Fçrster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from lownanomolar up to 10 mm,t hus covering aw ide range of biomolecular interactions.T he design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results. Single-moleculespectroscopyhasdevelopedintoapowerfulapproach for investigating biomolecular structure,d ynamics, and interactions,e specially in combination with Fçrster resonance energy transfer (FRET). [1] However,amajor challenge for studying the mechanisms of biomolecular interactions by single-molecule spectroscopy,s uch as FRET between two binding partners,isthat their affinities are often so low that they rapidly dissociate at the about 10 to 100 pm sample concentrations required for single-molecule detection. As ar esult, single-molecule studies with two fluorescently labeled interaction partners are often not possible at equilibrium. This limitation can be circumvented by forming the complex at high concentrations,r apidly diluting the sample to single-molecule concentrations,and monitoring its properties before dissociation. [2] However,there are currently no methods available that combine sufficiently large dilutions and short dead times to enable observation of such complexes before they dissociate.H erein, we demonstrate as olution to this problem using am icrofluidic device that enables the sample to be diluted more than 10 000-fold within milliseconds and allows the properties of the complex and its dissociation kinetics to be monitored by confocal singlemolecule spectroscopy.As uitable rapid dilution microfluidic device must meet several requirements.F irst, the dilution needs to be sufficiently rapid that even low-affinity complexes with high dissociation rates can be studied. Them inimum time for dilution in laminar flow is fundamentally limited by translational diffusion. Given the diffraction-limited size of the confocal observation volume and the diffusion coefficient of typical biomolecular samples,t he minimum dead time for a10000-to 100 000-fold dilution, for example,isinthe range of af ew milliseconds ( Figure S1 in the Supporting Information). Second, since not all applications require the same dilution factor,the device must provide dilutions that can be adjusted over aw ide range (about 1000-to 100 000-fold). Third, because high sample concentrations are required to form stable biomolecular complexes initially,t he device should have alow sample ...

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

Cited by 111 publications

References 61 publications

Mapping the conformation of a client protein through the Hsp70 functional cycle

Mapping the conformation of a client protein through the Hsp70 functional cycle

Recent advances in understanding catalysis of protein folding by molecular chaperones

Rapid Microfluidic Dilution for Single‐Molecule Spectroscopy of Low‐Affinity Biomolecular Complexes

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