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

Abstract

Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a “denaturation temperature” of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.

Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1

“…The fluorescence of this dye is quenched in hydrophilic and enhanced in hydrophobic environment. 37 8-Anilinonaphthalene-1-sulfonic acid (ANS) and its derivatives are widely used to detect Ca 2+induced conformational changes of EFCaBPs. 38 Yet, ANS binds to proteins not only by hydrophobic but also by electrostatic interactions.…”

“…37 Thus, the dye is more elongated and interacts specifically with hydrophobic regions. 37 It should be in general advantageous to detect the Ca 2+dependent accessibility of EFCaBPs' hydrophobic pockets. As a proof-of-principle, we showed enhanced fluorescence and thus binding of the dye to the hydrophobic patches of CaM, which become exposed in the presence of Ca 2+ .…”

Three Essential Resources to Improve Differential Scanning Fluorimetry (DSF) Experiments

“…Because DSF relies on the use of dye fluorescence as a proxy for protein unfolding, identifying and minimizing sources of protein-independent fluorescence is a critical step. Indeed, the fluorescence of SYPRO Orange is known to be sensitive to excipients that are common in biological buffers, such as glycerol, detergents, lipids and EDTA 34,43 . Here, we focus on two especially pernicious and common sources of protein-independent fluorescence: dye binding to colloidal aggregates and dye binding to the plastic used in manufacture of some microtiter plates.…”

“…Sometimes a validated ligand induces no ∆Tma, a replicated experiment produces different results, the data cannot be fit to a simple sigmoid, and so on. While excellent resources exist guiding applications of successful DSF, as well as some descriptions of its systematic shortcomings 33,34 , there is currently little to no framework for optimizing failed DSF conditions into successful ones, let alone how to do so efficiently. Instead, failures and solutions are passed on at conferences or group meetings if at all, so when an experiment fails, it is often unclear what to try next.…”

Poly(triazolyl methacrylate) glycopolymers as potential targeted unimolecular nanocarriers

“…Both fluorophores possess low quantum yields in aqueous media, resulting in a low fluorescence intensity, which increases in less polar environments. 36,37 Triazolyl methacrylate glycopolymers have been extensively investigated. 33,34,[38][39][40][41][42][43][44][45][46][47] Yet, to the best of our knowledge, this behaviour, suggestive of their surfactant-like properties and of their ability to non-covalently bind hydrophobic probes under aqueous conditions, has not been described.…”