Probing Protein–Chaperone Interactions with Single‐Molecule Fluorescence Spectroscopy

Hillger, Frank; Hänni, Dominik; Nettels, Daniel; Geister, Sonja; Grandin, Michelle; Textor, Marcus; Schuler, Benjamin

doi:10.1002/anie.200800298

Cited by 72 publications

(70 citation statements)

References 42 publications

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“…Under native conditions (Fig. 1B), two peaks are observed for each variant: the peaks at E ¼ 0.67 for the N variant, E ¼ 0.69 for the L variant (27), and E ¼ 0.98 for the C variant result from native rhodanese molecules; the peaks near E ¼ 0 result from molecules lacking an active acceptor dye and can be eliminated by dual color excitation of donor and acceptor (35,36) (Fig. 1B, see SI Appendix for details).…”

Section: Resultsmentioning

confidence: 99%

“…Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES.…”

mentioning

confidence: 99%

“…Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES.…”

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confidence: 99%

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Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

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113

124

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Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra-and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.I n the recent past, a large number of components have been identified that control and modulate protein folding in vivo. This machinery includes molecular chaperones (1-3), sophisticated quality control systems, and complex mechanisms for protein translocation and degradation (3, 4), reflecting the importance of regulating the delicate balance of protein folding, misfolding, and aggregation in the cell. Such cellular factors exert conformational constraints on protein molecules that are expected to have a strong effect on the corresponding free-energy surfaces for folding (5). However, while the combination of cellular, biochemical, and structural data has led to some plausible qualitative models for the processes involved, mechanistic investigations comparable to those of autonomous protein folding in vitro (5-8) have been complicated by the complexity of the systems and the conformational heterogeneity involved (9). Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES. However, the cavity is only slightly larger than the folded structure of typical proteins known to interact with the chaperonin. The large volume of unconfined unfolded protein chains compared to the size of the cavity raises the question of whether and how such strong confinement affects the folding reaction (12-16, 18, 31, 32). By labeling the classic substrate prote...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

Self Cite

113

124

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“…Here, we assume that the absorption and emission dipole moments are collinear for our dyes, so the fundamental anisotropy is given by r 0 = 0.4, close to the experimental value. 27 In the analysis of experimental data, often a doubleexponential decay is required to account for the contributions of rotational motion of both the fluorophore and the protein. 14,26,28 In the systems we investigate here, the proteins are large in comparison to the dyes and the measured dye rotational correlation times are one order of magnitude smaller than the expected rotational correlation time of the proteins.…”

Section: B Rotational Correlation Timementioning

confidence: 99%

Simulation of FRET dyes allows quantitative comparison against experimental data

Reinartz

Sinner

Nettels

et al. 2018

The Journal of Chemical Physics

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Fully understanding biomolecular function requires detailed insight into the systems' structural dynamics. Powerful experimental techniques such as single molecule Förster Resonance Energy Transfer (FRET) provide access to such dynamic information yet have to be carefully interpreted. Molecular simulations can complement these experiments but typically face limits in accessing slow time scales and large or unstructured systems. Here, we introduce a coarse-grained simulation technique that tackles these challenges. While requiring only few parameters, we maintain full protein flexibility and include all heavy atoms of proteins, linkers, and dyes. We are able to sufficiently reduce computational demands to simulate large or heterogeneous structural dynamics and ensembles on slow time scales found in, e.g., protein folding. The simulations allow for calculating FRET efficiencies which quantitatively agree with experimentally determined values. By providing atomically resolved trajectories, this work supports the planning and microscopic interpretation of experiments. Overall, these results highlight how simulations and experiments can complement each other leading to new insights into biomolecular dynamics and function.

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“…As an example, we use the stable complex of the protein rhodanese with the molecular chaperone GroEL. 37 Molecular chaperones are a class of proteins that assist other proteins during folding. 38 Topics of particular interest are the non-native structure and the conformational dynamics of rhodanese upon binding to GroEL, and the properties of the extremely stable rhodanese-GroEL complex.…”

Section: B Single Molecule Studiesmentioning

confidence: 99%

Scalable time-correlated photon counting system with multiple independent input channels

Wahl

Rahn

Röhlicke

et al. 2008

Review of Scientific Instruments

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Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information (time tagging), thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized to picoseconds of relative timing and yet operate independent of each other. This is achieved by means of a modular design with independent crystal-locked time digitizers and a central processing unit for sorting and processing of the timing data. The modules communicate through high speed serial links supporting the full throughput rate of the time digitizers. Event processing is implemented in programmable logic, permitting classical histogramming, as well as time tagging of individual photons and their temporally ordered streaming to the host computer. Based on the time-ordered event data, any algorithms and methods for the analysis of fluorescence dynamics can be implemented not only in postprocessing but also in real time. Results from recently emerging single molecule applications are presented to demonstrate the capabilities of the instrument.Scalable time-correlated photon counting system with multiple independent input channels Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information ͑time tagging͒, thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized ...

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Probing Protein–Chaperone Interactions with Single‐Molecule Fluorescence Spectroscopy

Cited by 72 publications

References 42 publications

Single-molecule spectroscopy of protein folding in a chaperonin cage

Single-molecule spectroscopy of protein folding in a chaperonin cage

Simulation of FRET dyes allows quantitative comparison against experimental data

Scalable time-correlated photon counting system with multiple independent input channels

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