EDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biophysical Chemistry

Background:

• Ethylenediaminetetraacetic acid (EDTA) is a common chelating agent in life sciences.
• EDTA can form supramolecular aggregates at pH > 8, potentially affecting assay results.
• SYPRO Orange is a fluorescent dye used in thermal shift assays (TSA) to detect protein stability.

Purpose of the Study:

• To investigate the cause of artifactual fluorescence signals observed in TSA when using EDTA as a buffer component at pH ≈ 10.
• To determine if interactions between EDTA and SYPRO Orange are responsible for the observed fluorescence changes.
• To characterize the nature of EDTA aggregates and their interaction with SYPRO Orange.

Main Methods:

• Thermal shift assays (TSA) with SYPRO Orange in the presence of EDTA and varying Ca2+ concentrations.
• Analytical ultracentrifugation to confirm the formation of EDTA aggregates.
• Molecular dynamics simulations to model the structure of EDTA aggregates and their interaction with SYPRO Orange.

Main Results:

• EDTA aggregates interact with SYPRO Orange at pH > 9 in a temperature-dependent manner, producing a false denaturation signal around 68°C.
• Titration of Ca2+ into SYPRO Orange and EDTA solutions quenched fluorescence, indicating ion-dependent interactions.
• Analytical ultracentrifugation and molecular dynamics simulations confirmed the formation of EDTA aggregates and proposed a structural model involving U-shaped EDTA4- in an inverse bilayer arrangement.

Conclusions:

• EDTA aggregates induce SYPRO Orange-based fluorescence signals in TSA, mimicking protein denaturation.
• These findings highlight the importance of considering potential interference between EDTA and fluorescent dyes in TSA.
• Future TSA experiments should ensure that results are not influenced by interactions between EDTA or related molecules and the fluorescent dye.