Fig 1.
Thermofluor assay signal in the absence of protein.
Fluorescence signal of SYPRO Orange in: H2O (yellow); H2O + 137 mM NaCl (green); H2O + 137 mM NaCl + 1.5 mM NaH2PO4 (blue); H2O + 137 mM NaCl + 1.5 mM NaH2PO4 + 2.7 mM KCl (dark blue); H2O + 137 mM NaCl + 1.5 mM NaH2PO4 + 2.7 mM KCl + 1.5 mM KH2PO4 (purple); H2O + 137 mM NaCl + 1.5 mM NaH2PO4 + 2.7 mM KCl + 1.5 mM KH2PO4 + 100 mM EDTA (red); the signals were normalized to the overall highest detected signal.
Fig 2.
Influence of EDTA and EGTA on the SYPRO Orange-based fluorescence in a Thermofluor assay.
(A) Dependence of the relative change in fluorescence intensity on the EDTA concentration (pH = 10); EC50 = 36.3 mM EDTA. (B) Dependence of the relative change in fluorescence intensity of the SYPRO Orange/EDTA system on the pH (100 mM EDTA); half-maximal change of fluorescence at pH = 9.9. (C) Dependence of the relative change in fluorescence intensity of the SYPRO Orange/EDTA system on the addition of Ca2+ (pH 10). Circles represent results for a sample in the presence of 100 mM EDTA, EC50 = 100 mM Ca2+; squares represent results for a sample in the absence of EDTA (negative control). (D) Dependence of the relative change in fluorescence intensity of the EGTA–SYPRO Orange system (pH = 10) on EGTA concentration and the absence or presence of Ca2+. (A)-(D): Values are normalized with respect to the minimal (0%) and maximal (100%) change detected. The error bars show the SEM. *: p < 0.0001.
Fig 3.
MD simulations to investigate EDTA aggregation and interaction with SYPRO Orange.
(A) SASA and frequency distribution of the SASA over all 20 EDTA molecules in the simulation box; EDTA4- (black), EDTA3- (red), the arrow shows which snapshot was used to visualize the EDTA aggregate. (B) Snapshot of an MD trajectory showing an EDTA4—Na+ aggregate; the snapshot was taken at 500 ns (see arrow in panel A); EDTA4-: green: C atoms, blue: N atoms, red: O atoms; magenta: Na+ ions. Box: This part of the image was zoomed to get a better view on the bilayer within the aggregate. For clarity, some EDTA molecules in front of the bilayer were discarded. (C) Zoomed view on an EDTA4—Na+ aggregate showing a bilayer formation.
Fig 4.
Structural model from MD simulations of EDTA aggregates and SYPRO Orange forming a complex with the hydrophobic region of EDTA.
(A) Average number of EDTA molecules within 5 Å of the core region of SYPRO Orange for EDTA4- (black) and EDTA3- (red). The core region is defined as the two aromatic rings with the linker (see S1 Fig). (B) Frequency distribution of the distance of the center of the ethylene group of every EDTA4- molecule to the center of mass of the core region (see panel A) of SYPRO Orange. (C) Frequency distribution of the distance of the center of the ethylene group of every EDTA3- molecule to the center of mass of the core region (see panel A) of SYPRO Orange. Individual distance distributions are depicted in S10 Fig. The force field ff12SB was applied in both MD simulations. (D) Snapshot of an MD trajectory showing the EDTA4—Na+ aggregate from Fig 3B (surface representation) to which two SYPRO Orange molecules bind non-covalently. The coloring of the surface is according to the local partial atomic charge. (E) Schematic figure showing the association of SYPRO Orange with an EDTA4—Na+ aggregate as derived from panel D. The two non-polar surfaces of the aggregate and SYPRO Orange are highlighted by dashed borders.