Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be af...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2017-01, Vol.89 (1), p.694-702
Main Authors: Kempe, Daryan, Cerminara, Michele, Poblete, Simón, Schöne, Antonie, Gabba, Matteo, Fitter, Jörg
Format: Article
Language:English
Subjects:
Analytical chemistry
Aqueous solutions
Buffers
Deviation
DNA - chemistry
Fluorescence
Fluorescence Resonance Energy Transfer - methods
Fretting
Glycerol - chemistry
Guanidine - chemistry
Kinases
Oligodeoxyribonucleotides - chemistry
Physical properties
Reagents
Sodium chloride
Sodium Chloride - chemistry
Solutions
Solvents
Static Electricity
Viscosity
Water - chemistry
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Summary:The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye–dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil–globule transition.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b03147