Measuring Surface Binding Thermodynamics and Kinetics by Using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

Thompson, Nancy L.; Navaratnarajah, Punya; Wang, X.

doi:10.1021/jp1069708

Cited by 13 publications

(23 citation statements)

References 47 publications

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“…As previously shown, 35 the probability that a bleached molecule that dissociates from the origin at time zero has rebound at least once within the illuminated and observed area after the duration of the postbleach observation is…”

Section: Resultsmentioning

confidence: 65%

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

et al. 2011

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The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor’s affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s−1, respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.

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Section: Resultsmentioning

confidence: 65%

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

et al. 2011

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“…30 , 31 For the reversible binding of molecules at a surface, these dynamic processes can include diffusion in and out of the observation volume and absorption and desorption of the molecules to the surface. 30 − 32 The surface specificity of SHCS eliminates the contributions from diffusion of molecules in solution, such that the only contributing factors to the correlated fluctuations in the SH signal are from the surface binding kinetics. 29 …”

Section: Methodsmentioning

confidence: 99%

Determination of Multivalent Protein–Ligand Binding Kinetics by Second-Harmonic Correlation Spectroscopy

Sly

Conboy

2014

Anal. Chem.

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Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 106 M–1·s–1 at 0.43 μM PnA to (1.1 ± 0.1) × 105 M–1·s–1 at 12 μM PnA. CTB–GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 109 M–1·s–1 at 0.5 nM CTB to (3.5 ± 0.2) × 106 M–1·s–1 at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 109 M–1 and (2.8 ± 0.5) × 1013 M–1, respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB–GM1 demonstrates positive ligand–ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein–ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

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“…The resulting contrast and detectability of SHG nanoprobes provide a unique alternative for fluorescence imaging in vivo (reviewed by Dempsey et al (2012b)). For example, the use of SHG nanoprobes in second harmonic generation correlation spectroscopy (SHG-CS) would offer an effective method for the precise detection of single molecule dynamics over extended periods of time with up to a 100-fold lower detection limit than that of conventional FCS (Liu and Irudayaraj, 2013), whose analysis is often complicated by background fluorescence and photobleaching (Hansen and Harris, 1998;Thompson et al, 2011). Additionally, different SHGcapable materials have distinct spectral SHG signal intensity profiles and offer the possibility of detecting multiple SHG nanoprobe-labeled proteins of interest in parallel.…”

Section: Single Molecule Sensitivity With Engineered Nanoprobesmentioning

confidence: 99%

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

2015

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In recent years, advances in imaging probes, cutting-edge microscopy techniques and powerful bioinformatics image analysis have markedly expanded the imaging toolbox available to developmental biologists. Apart from traditional qualitative studies, embryonic development can now be investigated in vivo with improved spatiotemporal resolution, with more detailed quantitative analyses down to the single-cell level of the developing embryo. Such imaging tools can provide many benefits to investigate the emergence of the asymmetry in the early mammalian embryo. Quantitative single-cell imaging has provided a deeper knowledge of the dynamic processes of how and why apparently indistinguishable cells adopt separate fates that ensure proper lineage allocation and segregation. To advance our understanding of the mechanisms governing such cell fate decisions, we will need to address current limitations of fluorescent probes, while at the same time take on challenges in image processing and analysis. New discoveries and developments in quantitative, single-cell imaging analysis will ultimately enable a truly comprehensive, multidimensional and multi-scale investigation of the dynamic morphogenetic processes that work in concert to shape the embryo.

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Measuring Surface Binding Thermodynamics and Kinetics by Using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

Cited by 13 publications

References 47 publications

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

Determination of Multivalent Protein–Ligand Binding Kinetics by Second-Harmonic Correlation Spectroscopy

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

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