The Characterization of Biomolecular Interactions Using Fluorescence Fluctuation Techniques

Margeat, Emmanuel; Boukari, Hacène; Royer, Catherine A.

doi:10.1007/978-0-387-35966-3_1

Cited by 4 publications

(11 citation statements)

References 123 publications

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“…In this method, two dyes within the sample are simultaneously excited by means of a 2PE laser (TPFCCS) [62], two 1PE lasers [63] or a single 1PE laser (SWFCCS) [64], and the emission of the two fluorophores is detected in two separate channels. This procedure allows the generation of two autocorrelation functions, one for each dye, and a cross-correlation curve, which contains information on the diffusion and concentration of the complexes in which both labels diffuse together [26,63]. Under ideal conditions with no cross-talk, i.e.…”

Section: Fluorescence Correlation and Cross-correlation Spectroscopymentioning

confidence: 99%

“…FCS experiments allow monitoring any dynamic event that provokes measurable changes in the intensity emitted by the fluorescent molecules within a tiny open volume element created in a solution, membrane or cell by a focused laser beam [26,60]. The most evident source of fluorescence intensity fluctuations is the translational Brownian motion of the molecules in and out of the detection volume.…”

Section: Fluorescence Correlation and Cross-correlation Spectroscopymentioning

confidence: 99%

“…Because of these observations, biophysicists have become particularly interested in investigating the energetic and kinetic parameters underlying biological reactions, in the physiological environments in which they take place. Indeed, the identification and quantification of biomolecules and their functional complexes inside the living cells is nowadays feasible, due to the recent development of biological applications of single molecule fluorescence methods [26][27][28][29]. However, quantitative determinations inside cells are still challenging and hence, detailed in vitro studies remain necessary as a first step to approach biological questions.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Zorrilla

Lillo²

2009

CPPS

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Fluorescence spectroscopy methods have been proved to be powerful tools for the quantitative investigation of homologous and heterologous interactions, rotational and translational diffusion, and structural dynamics of biological molecules in crowded media. In addition to their high sensitivity, these methods present the advantage that the selective fluorescent labeling of the biomolecules under study allows distinguishing them from the background species. Moreover, the recent development of biological applications of single molecule fluorescence micro-spectroscopy methods has opened the possibility of performing quantitative determinations inside cells. In the last decades, theoretical and experimental studies have demonstrated the possible influence of the high concentration of macromolecules within living systems, on the thermodynamics and kinetics of biological reactions. Therefore, there is a growing interest in quantitatively evaluating the interactions involving biomolecules in the natural environment in which they occur. Since this is not always feasible, experiments conducted in model crowded conditions, resembling physiological media, may contribute to reduce the gap between traditional in vitro and in vivo experiments. In this review we will discuss the application of some fluorescence spectroscopy approaches, for the identification and quantification of biological macromolecules and their functional interactions in model crowded conditions.

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Section: Fluorescence Correlation and Cross-correlation Spectroscopymentioning

confidence: 99%

Section: Fluorescence Correlation and Cross-correlation Spectroscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Zorrilla

Lillo²

2009

CPPS

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“…In an FCS experiment, a laser beam is sharply focused into the solution, generating an open volume element, and the fluctuations of the fluorescence emitted by the molecules within the volume are detected by an avalanche photodiode. The intensity profiles recovered are used to calculate autocorrelation functions, G (τ), which contain information on the translational diffusion of the fluorescent molecules as well as on their concentration in the solution 14,15 …”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

“…Although the fundamentals of FCS methods have been well known for years, the application of this single‐molecule approach to the physical–chemical analysis of solutions containing biomolecules is relatively recent 15,16 . Researchers working on FCS are in general aware of how to avoid common artifacts, such as excitation saturation and photobleaching, which have already been extensively discussed elsewhere 16,17 .…”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

Investigating Transcriptional Regulation by Fluorescence Spectroscopy, from Traditional Methods to State‐of‐the‐Art Single‐Molecule Approaches

Zorrilla

Lillo

Chaix

et al. 2008

Annals of the New York Academy of Sciences

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Gene expression regulation, in particular at the level of transcription, has been demonstrated to play a key role in the development of human diseases, including cancer, and in bacteria it is crucial for proliferation as well as for pathogenicity. Transcriptional regulation is based on complex networks of interactions, including those of the regulatory proteins with the operator DNAs, which are further modulated by ligands. Thus, understanding transcriptional regulation mechanisms requires a thorough analysis of the physical parameters underlying the interactions involved. Among the panoply of methods available, fluorescence spectroscopy-based approaches have been widely used for the assessment of the thermodynamics and structural dynamics of biomolecular interactions. Here we will discuss the application of three fluorescence spectroscopy methods--fluorescence anisotropy and fluorescence correlation and cross-correlation spectroscopy--for the investigation of protein-DNA, protein-protein, and protein-ligand interactions. The weaknesses and the strengths of each method will be highlighted on the basis of our experience in the analysis of the interactions of bacterial repressors implicated in transcriptional regulation in bacilli.

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Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

Zhao

Ingaramo

et al. 2014

Anal. Chem.

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Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work, we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for subdiffraction microscopy and enables FDS-SV studies of their interactions and size distributions. At the same time, we find that conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the signal quality.

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The Characterization of Biomolecular Interactions Using Fluorescence Fluctuation Techniques

Cited by 4 publications

References 123 publications

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Investigating Transcriptional Regulation by Fluorescence Spectroscopy, from Traditional Methods to State‐of‐the‐Art Single‐Molecule Approaches

Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

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