Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules

Doose, Sören; Neuweiler, Hannes; Sauer, Markus

doi:10.1002/cphc.200900238

Cited by 449 publications

(475 citation statements)

References 100 publications

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“…The first component captures mainly an increase in the molecular brightness over time ( Fig. 3 A, B, S6-8), which is probably caused by the burial of tryptophan residues in the native structure that quench donor and acceptor in the denatured state (40). The second component corresponds to the changes in all other spectroscopic parameters, e.g., the transfer efficiency ( Fig.…”

Section: Resultsmentioning

confidence: 98%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

109

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: 98%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

109

124

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“…It was mainly caused by intermolecular self-absorption, fluorescence quenching, and energy conversion of collision at high concentration. [18][19][20][21][22] APBA was used in modification to endow HPAMAMs with glucose-sensitive property. Phenylboronic acid has been widely utilized for the design of chemosensors in the detection of saccharides over the past decades.…”

Section: Resultsmentioning

confidence: 99%

Facile one-pot synthesis of fluorescent hyperbranched polymers for optical detection of glucose

Jiang

Wang

et al. 2014

Designed Monomers and Polymers

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Fluorescent and glucose-sensitive hyperbranched poly(amidoamine)s (HPAMAMs-B) were synthesized via Michael addition of 1-(2-aminoethyl) piperazine, methyl acrylate, and 3-aminobenzeneboronic acid (APBA). These hyperbranched polymers emitted chartreuse photoluminescence and showed emission band at 378 nm under excitation wavelength. The intensity of emission band was enhanced with increasing the amount of APBA in HPAMAMs-B. The glucose sensitivity of HPAMAMs-B has been investigated at different glucose concentrations and pH conditions. These hyperbranched polymers showed potential for biomedical applications such as fluorescence imaging technology and glucose detecting sensors.

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“…[7][8][9] Furthermore, their application and use in cell biology demands not only detailed knowledge about toxicity but also about potential interactions with various biochemical compounds that might influence fluorescence emission. Whereas such influences on organic dyes have been described in literature, [10] to our knowledge, systematic investigations of the influence of nucleotides and amino acids on the fluorescence emission of QD are yet to be studied. In current literature, quenching of QD by metal ions, such as Cu 2þ and Ag þ , and by organic molecules containing amino-groups have been reported.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Quenching of Quantum Dots by DNA Nucleotides and Amino Acids

Siegberg¹,

Herten²

2011

Aust. J. Chem.

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Quantum dots found widespread application in the biosciences as bright and highly photo-stable fluorescent probes, i.e. for single-particle tracking. In this work we used ensemble spectroscopy and single-molecule techniques to study the quenching of quantum dots by various biochemical compounds that are usually present in living cells and might thus influence the experiments. We found not only nucleotides such as cytosine, guanine, and thymine can significantly influence the fluorescence emission of CdSe and CdTe quantum dots, but also amino acids, like asparagine and tryptophan. Bulk studies on fluorescence quenching indicated a static quenching mechanism. Interestingly, we could also show by single-molecule fluorescence spectroscopy that quenching of the quantum dots can be irreversible, suggesting either a redox-reaction between quantum dot and quencher or strong binding of the quencher to the surface of the bio-conjugated quantum dots.

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Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules

Cited by 449 publications

References 100 publications

Single-molecule spectroscopy of protein folding in a chaperonin cage

Single-molecule spectroscopy of protein folding in a chaperonin cage

Facile one-pot synthesis of fluorescent hyperbranched polymers for optical detection of glucose

Fluorescence Quenching of Quantum Dots by DNA Nucleotides and Amino Acids

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