Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions

Area of Science:

• Biophysics
• Physical Chemistry

Background:

• Chemical denaturants, salts, and crowding agents alter buffer solution properties like viscosity and dielectric constant.
• These solvent property changes can impact the accuracy of biophysical measurements, particularly single-molecule Förster Resonance Energy Transfer (smFRET).

Purpose of the Study:

• To systematically evaluate the influence of solvent characteristics on smFRET data.
• To develop and validate a method for accurate smFRET analysis in diverse buffer conditions.
• To investigate the coil-globule transition of proteins using smFRET.

Main Methods:

• Development of a confocal microscopy-based method for rapid determination of fluorescence quantum yields at sub-nanomolar concentrations.
• Analysis of smFRET data from DNA oligonucleotides in various buffer solutions (aqueous, glycerol, guanidine hydrochloride, sodium chloride).
• Calculation of interdye distances and assessment of electrostatic dye-dye repulsion effects.

Main Results:

• Solvent-induced spectral and photophysical changes in fluorescent labels can cause up to 4 Å deviations in calculated interdye distances if not considered.
• Electrostatic dye-dye repulsions are negligible for distances greater than 50 Å.
• The study validated the coil-globule transition mechanism in phosphoglycerate kinase (PGK) using the developed smFRET approach.

Conclusions:

• Accurate smFRET analysis requires accounting for solvent effects on fluorophore photophysics.
• The developed method provides a robust approach for characterizing smFRET samples and analyzing smFRET data in complex solutions.
• This work enhances the understanding of protein folding dynamics and the impact of solution conditions on biomolecular structure determination.