Structural relaxation of rhodamine dyes with different N-substitution patterns: A study of fluorescence decay times and quantum yields

Vögel, Martin; Rettig, Wolfgang; Sens, Rüdiger; Drexhage, K. H.

doi:10.1016/0009-2614(88)85007-3

Cited by 175 publications

(153 citation statements)

References 32 publications

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“…The mechanism by which TMR senses changes in its environment is related to polarity, the fluorescence quantum yield and lifetime increasing with decreasing polarity. 47, 48 The longest reported lifetime for TMR in apolar media is 3.7 ns, a value close to the mean lifetime of ligand-free β 2 AR-TMR measured here (3.4 ns). We observe, however, an even longer mean lifetime of 4.4 ns for agonist-bound β 2 AR-TMR, incompatible with a solely polarization-related mechanism.…”

Section: Discussionsupporting

confidence: 81%

Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

et al. 2011

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G Protein-Coupled Receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β2-Adrenergic Receptors (β2ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently-labeled β2AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β2ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with stretching exponent β ~ 0.5, suggesting protein dynamics over a range of timescales.

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Section: Discussionsupporting

confidence: 81%

Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

et al. 2011

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“…We used BIFL to study the dynamics of a TMR-labeled oligodeoxynucleotide (CAA AGC GCC ATT CGC CAT TC) in aqueous solution. TMR is ideal for an environmental probe because of two properties: (i) the polarity of the environment influences its fluorescence lifetime, which ranges from 2.4 ns in aqueous solution to 3.6 ns in nonpolar solvents (15,22,32); (ii) the dye characterizes the binding site on the DNA, because its fluorescence is specifically quenched by guanine (33).…”

Section: Resultsmentioning

confidence: 99%

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy

Eggeling

Fries

Brand

et al. 1998

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

341

338

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Laser-induced fluorescence detection of single fluorescent molecules represents the ultimate level of sensitivity for fluorescence-based assays in analytical chemistry, biology, and medicine (1-14). Various groups have achieved singlemolecule detection in solution (1-6) and at surfaces (7-10).Recently it was possible to characterize single molecules in solution by their fluorescence emission spectra (12) or fluorescence lifetimes (13,14). Single-molecule spectroscopy eliminates ensemble averaging and allows individual members of a population to be probed, thereby yielding more direct information regarding the distribution of molecular and kinetic properties. This constitutes a major step toward the study of single-molecule dynamics and molecular recognition in solution. The recent improvements in single-molecule detection lead us to expect many diagnostic applications for such measurement techniques in the near future, such as monitoring enzyme function and conformational dynamics of DNA.An increasing number of publications (15-19) employing fluorophores covalently linked to polynucleotide sites illustrate the widespread interest in investigating thermodynamic, kinetic, and structural characteristics of various DNA molecules. Yet the fluctuations of the conjugated fluorophore between several conformational states, each with a different fluorescence quantum yield (15, 16), introduces a large degree of uncertainty to such evaluations.Rigler and coworkers recently investigated the structural dynamics measurements of a tetramethylrhodamine (TMR)-labeled DNA duplex in solution (16) and immobilized on a surface (20). They performed classical ensemble and singlemolecule measurements using fluorescence correlation and time-resolved fluorescence spectroscopy that were completed by time-integrated fluorescence decay measurements of single-molecule transits. These authors proposed a millisecond equilibrium between two conformational states characterized by different photobleaching lifetimes, fluorescence lifetimes of 0.9 and 3.7 ns, and a widely distributed conformational relaxation rate constant obeying a stretched exponential law, which indicates the existence of numerous conformational substates (21).So far, conventional confocal spectroscopic techniques (1, 16) have been used to analyze several fluorescence bursts caused by individual molecules diffusing through the microscopic, open measurement volume at once, thereby resulting in an averaging of properties over the single-molecule population under analysis. Fluorescence decay curves have been measured for single molecules (16,22), but the signal was integrated over the entire observation time, resulting in a loss of the kinetic information. Therefore, we have developed an experimental technique for specific burst analysis that avoids the inclusion of extraneous events and registers the macroscopic detection time of each single photon event for subsequent kinetic analysis.In this paper, we describe the identification and detection of different temporally resolved conf...

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“…However, this process does not produce another emissive species, but leads to the population of an entirely non-emissive species, most likely connected to the concept of twisted intramolecular chargetransfer (TICT) states. [47] Support is also furnished by investigations of solute-solvent complexes [21] or squaraines that are pretwisted, because of the introduction of bulky substituents in ortho position to the b1,b1' bonds, [26] both of which revealed decreased fluorescence quantum yields.…”

Section: Wwwchemeurjorgmentioning

confidence: 99%

Squaraines as Reporter Units: Insights into their Photophysics, Protonation, and Metal‐Ion Coordination Behaviour

Ros‐Lis

Martínez‐Máñez

Sancenón

et al. 2008

Chemistry A European J

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The synthesis, photophysical properties, protonation, and metal-ion coordination features of a family of nine aniline-based symmetrical squaraine derivatives are reported. The squaraine scaffold displays very attractive photophysical properties for a signalling unit. These dyes show absorption and weakly Stokes-shifted, mirror-image-shaped emission bands in the visible spectral range and there are no hints of multiple emission bands. The mono-exponential fluorescence decay kinetics observed for all the derivatives indicate that only one excited state is involved in the emission. These data stress the interpretation that squaraines can be regarded as polymethine-type dyes. From a coordination chemistry point of view, the squaraines possess four potential binding sites; that is, two nitrogen atoms from the anilino groups and two oxygen atoms from the central C(4)O(2) four-membered ring. These coordination sites are part of a cross-conjugated pi-system and coordination events with protons or certain metal ions affect the electronic properties of the delocalised pi-system dramatically, resulting in a rich modulation of the colour of the squaraines. The absorption band at around 640 nm is blue-shifted when coordination at the anilino nitrogen atoms occurs, whereas coordination to the C(2)O(4) oxygen atoms results in the development of red-shifted bands. Addition of more than one equivalent of protons or metal cations could additionally entail mixed N,O- or N,N-coordinated complexes, manifested in the development of a broad band at 480 nm or complete bleaching in the visible range, respectively. Analysis of the spectrophotometric titration data with HYPERQUAD yielded the macroscopic and microscopic stability constants of the complexes. Theoretical modelling of the various protonated species by molecular mechanics methods and consideration of some of the title dyes within the framework of molecular chemosensing and molecular-scale "logic gates" complement this contribution.

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Structural relaxation of rhodamine dyes with different N-substitution patterns: A study of fluorescence decay times and quantum yields

Cited by 175 publications

References 32 publications

Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy

Squaraines as Reporter Units: Insights into their Photophysics, Protonation, and Metal‐Ion Coordination Behaviour

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