Fluorescence quenching: A tool for single-molecule protein-folding study

Zhuang, Xiaowei; Ha, Taekjip; Kim, Harold D.; Centner, Thomas; Labeit, Siegfried; Chu, Steven

doi:10.1073/pnas.97.26.14241

Cited by 186 publications

(158 citation statements)

References 28 publications

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“…Upon the addition of GdnHCl, the fluorescence intensity of the molecules increased abruptly and significantly, and the number of fluorescent particles visible increased (Fig. 1a Lower), similarly to the observations of Zhuang et al (26). When GdnHCl was injected in the microchamber in steps of increasing concentration, the fluorescence of the same titin molecule increased in a stepwise fashion; at 7 M GdnHCl, its intensity was about four times that observed in the absence of the denaturant (Fig.…”

supporting

confidence: 70%

“…Individual titin molecules can be made visible under aqueous conditions after labeling with fluorophores. Fluorescently labeled titin molecules have recently been studied under various conditions: equilibration to a surface (21-23), stretching with meniscus force (22, 23) and direct manipulation (24), and chemical denaturation (25,26). Chemical denaturation has been shown to cause an abrupt and significant rise in the fluorescence intensity of Oregon Green 488 (Molecular Probes) maleimide-labeled titin molecules, which has been interpreted to derive from abolishing the fluorescence selfquenching present in the native state of the protein (26).…”

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confidence: 99%

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Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Grama

Somogyi

Kellermayer

2001

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

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The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding͞refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.T itins are 3.0-3.7 MDa filamentous proteins extending between the Z-and M-lines of the sarcomere (1-4). Titin is a tandem array of Ig type C2 and fibronectin (FN) type III domains (Ϸ300 per molecule) interspersed with unique sequences, most notably the Pro-, Glu-, Val-, and Lys-rich PEVK segment, and the N2A and N2B segments (5). The I-band segment of titin acts as a molecular spring, whose elastic properties define the passive or restoring mechanical properties of striated muscle (6-9). By contrast, titin's A-band segment is thought to function as a scaffold that defines structural regularity within the A band (10). Recent single-molecule experiments often have used titin as a model system because of a large size that makes it relatively easily accessible to such investigations. Single-molecule-mechanics experiments using either optical tweezers (11-13) or atomic force microscopy (AFM; ref. 14) described titin as an entropic chain in which domain unfolding occurs at high forces and refolding occurs at low forces. These experiments formed the basis of many theoretical and modeling works that addressed the mechanisms of mechanically driven protein folding (15)(16)(17)(18)(19)(20). Individual titin molecules can be made visible under aqueous conditions after labeling with fluorophores. Fluorescently labeled titin molecules have recently been studied under various conditions: equilibration to a surface (21-23), stretching with meniscus force (22, 23) and direct manipulation (24), and chemical denaturation (25,26). Chemical denaturation has been shown to cause an abrupt and significant rise in the fluorescence intensity of Oregon Green 488 (Molecular Probes) maleimi...

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supporting

confidence: 70%

mentioning

confidence: 99%

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Grama

Somogyi

Kellermayer

2001

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

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“…190 Labeling of 85% of the cystein residues with Oregon Green 488 did not modify the folding properties of the protein, as verified by single-molecule force spectroscopy on molecules adsorbed on a gold surface, using an atomic force microscope. 193 A sawtooth pattern with periodicity of ∼25 nm was observed as expected in the force vs extension curves for 40% of the labeled titin molecules, matching that observed for the same proportion of unlabeled proteins.…”

Section: Self-quenching Of Identical Fluorophores-zhuang Et Al Firstmentioning

confidence: 99%

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet¹,

Weiss²,

Jaeger³

2006

ChemInform

125

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“…With rapidly advancing fluorescence detection and microscopy capabilities, single-molecule studies are expanding to address questions related to structure and function in many biological systems, including enzymes (16), molecular motors (17,18), transcription (19,20), and protein and RNA dynamics (21)(22)(23)(24). Fluorescence resonance energy transfer (FRET) measurements have been used to track the conformational changes of single RNA or protein molecules, identify intermediates in RNA folding, and examine RNA-protein interactions (25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme

Xie

Srividya²,

Sosnick³

et al. 2004

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

121

115

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The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I eq-to-N, and focused on the Ieq-to-N transition. The present study focuses on the U-to-I eq transition. T he relationship between primary sequence and 3D structure is a fundamental issue in macromolecular folding (1, 2). Compared with the two-state folding behavior generally seen with small proteins (3, 4), RNA folding often involves metastable intermediates (5-9). This difference is attributed, in part, to the weakness of the protein interactions relative to the nucleic acid interactions, such as base pairing and stacking. Therefore, the free energy landscape for RNA folding is punctuated with deeper basins with populated folding intermediates (10-12). These intermediates can be populated under equilibrium, governed by thermodynamic (solvent and temperature) conditions, or transiently populated during nonequilibrium folding (13).The catalytic domain (C-domain) of Bacillus subtilis P RNA is a good model system to address RNA folding because it folds without kinetic traps and is large enough to exhibit the folding phenomenology of large RNAs (14). A minimalist folding pathway, derived from ensemble experiments performed as a function of Mg 2ϩ concentration, includes an unfolded state (U), a populated intermediate state (I eq ), and a native state (N). I eq is the thermodynamic reference state that establishes the stability of N; it represents the most populated species preceding N. The U-to-I eq transition, occurring at Mg 2ϩ concentrations Ϸ10-fold lower than that required for the I eq -to-N transition, has been proposed to consist of multiple transitions that were not resolved in ensemble measurements (14). Elucidation of the structural and dynamic characteristics of these intermediates is crucial for understanding the folding of large RNA in general.Single-molecule measurements look beyond ensemble averages to obtain novel and complementary information, as first demonstrated for ion channels (15). With rapidly advancing fluorescence detection and microscopy capabilities, single-molecule studies are expanding to address questions related to structure and function in many biological systems, including enzymes (16), molecular motors (17, 18), transcription (19,20), and protein and RNA dynamics (21-24). Fluorescence resonance energy transfer (FRET) measurements have been used to track the conformational changes of single RNA or protein molecules, identify intermediates in RNA folding, and examine RNA-protein interactions (25)(26)(27)(28)(29).The objective of the present single-molecule FRET study is to further elucidate the U-to-I eq transition in the equilibrium folding of the C-domain of P RNA. Folding intermediates are revealed by investigating the single-molecule FRET efficiency (E FRET ) distributions as a function of Mg 2ϩ concen...

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Fluorescence quenching: A tool for single-molecule protein-folding study

Cited by 186 publications

References 28 publications

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme

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