Spectroscopy data of ceftriaxone-lysozyme interaction and computational studies

The data article presents the results obtained from fluorescence and synchrotron radiation circular dichroism spectroscopies about the lysozyme-ceftriaxone interaction at neutral and acidic pH values as well as the computational calculations described in the accompanying research article (Ruzza et al., sub) [1].


Type of data
The potential thermal and UV stabilizing effects of ceftriaxone on HL and HEWL were monitored as complementary methods to determine ceftriaxone-lysozyme binding interactions.

Data accessibility
All referenced data is in the article.

Value of the data
Data enlarges the panel of available anti-amyloidogenic molecules acting on lysozyme Data expands the array of known amylodogenic targets of ceftriaxone, a widely-used antibiotic Data provides new references for characterization of anti-amyloidogenic compounds

Data
Evaluation of ceftriazone Ceftriaxone (Cef, CAS number 104376-79-6) on PC12 apoptosis using 6-OHDA cell toxicity assay ( Fig. 1) was identified [2]. The influence of pH values on the monomeric or oligomeric states of lysozyme was evaluated by native PAGE (Fig. 2). Fluorescence spectroscopy measurements were used to determine the dissociation constant (K d ) (Figs. 3A and 4A) using a nonlinear regression analysis [3]. Fluorophorus populations: one accessible to the quencher and the other inaccessible (see Table 1 in Ref. [1]) was determined by The Stern-Volmer analysis [4] of the Trp emission quenching (Figs. 3B and 4B). The influence of Cef on lysozyme stability was evaluated by both UV and thermal denaturation assays using synchrotron radiation circular dichroism (Figs. [5][6][7][8][9]. Hydrodynamic radii of the protein in the presence and absence of Cef were measured using dynamic light scattering (Fig. 10). Computational calculations were carried out as described in the methods (Figs. 11 and 12, Tables 1-3).

Synchrotron radiation circular dichroism (SRCD)
HEWL, recombinant HL and Cef were purchased from Sigma-Aldrich and used without any further purification. Proteins (0.5 mg/mL) were dissolved either in 20 mM phosphate buffer, pH 6.8, or in 70 mM glycine-HCl/80 mM NaCl buffer, pH 2.7. Cef stock solutions were prepared in the same buffers. Sample concentrations were determined by UV-vis spectroscopy. SRCD spectra from 180 to 260 nm were collected at Diamond B23 beamline module end-station B using integration time of 1 s, 1 nm digital resolution, 39 nm/min scan speed and 1.8 or 1.2 nm bandwidth according to the experiments. Spectra were measured using Suprasil cell (Hellma Ltd.) with 0.02 cm path length filled with 60 µL of solution. Thermal stability was monitored in the 5-90°C temperature range at 5°C increments with 5 min equilibration time using Quantum Peltier temperature controller. Protein UV photo-denaturation was investigated measuring twenty-five consecutive repeated scans for each sample. SRCD spectra were processed and analyzed using CD Apps software [6].

Secondary structure estimations (SSE)
SSE made from SRCD data collected at Diamond Light Source through CDApps software [6] using the CONTILL [6] algorithm.       7. Secondary structure content versus temperature. Plot of β-strand and unordered content for the HEWL alone (black, pH 6.8; blue, pH 2.7) and in presence of 2 eq. of ceftriaxone (red, pH 6.8; magenta, pH 2.7) determined with CONTINLL [5] of CDApps [6] from SRCD data versus temperature.     For each complex, the protein residues forming contacts with Cef atoms within 5 Å are shown in black, while the corresponding residues of the other protein are reported in gray. In both sequences, red bold, red and dark orange characters indicate major, non-conservative and conservative mutations, respectively. Light yellow areas enclose residues forming hydrophobic patches/pockets in which the ligand docks in the pose under examination; cyan lines, blue lines and green two connected circles show H-bonds involving protein backbone, H-bonds involving protein sidechain, and π-stacking interaction, respectively. For multiple H-bonds, the number of interactions is shown on the left of the corresponding symbol.

Table 2
Main interactions and calculated binding free energy (ΔG b ) in models of the HEWL-Cef complexes at different pHs. "q" field contains the net ligand charge. Protein residues are considered interacting with Cef when at least one atom of the residue is within 5 Å of at least one ligand atom. "Special interactions" field includes H-bond (HB, normal style); HB reinforced by ionic interactions (HB-II, bold); π stacking (italics); positive charge-π interaction (underlined italics). NHbb and CObb indicate backbone peptide hydrogen and oxygen atoms, respectively. The SEM for ΔG b values is reported.
pH Complex a q (a.u.) HEWL residues interacting with Cef Special interactions (HB/HB-II/stacking/pos. charge-π)   Table 3 Main interactions and calculated binding free energy (ΔG b ) in models of the HL-Cef complexes at neutral pH. "q" field contains the net ligand charge. Protein residues are considered interacting with Cef when at least one atom of the residue is within 5 Å of at least one ligand atom. "Special interactions" field includes H-bond (HB, normal style); HB reinforced by ionic interactions (HB-II, bold); π stacking (italics); positive charge-π interaction (underlined italics

Fluorescence spectroscopy
Fluorescence emission spectra were recorded on a Perkin-Elmer LS-50B spectrofluorimeter using emission and excitation slit widths of 2.5 nm, at 25°C, subtracting the buffer background and correcting for dilution. Excitation wavelength was set to 295 nm [4] and the emission wavelengths were scanned from 305 nm to 475 nm in 1 nm increments. The fluorescence intensity were corrected for absorption of exciting light and reabsorption of emitted light [8], while the Trp fluorescence quenching as well as the Cef affinity were examined by the Stern-Volmer equation and a nonlinear regression respectively (see Material and Methods in accompanying article [1]).

in vivo studies
in vivo studies were conducted as described in Ref. [2].

Dynamic light scattering
Solutions of HL 0.5 mg/mL in Gly-HCl saline buffer, at either pH 2.7 or 6.8, was prepared with 0.03% NaN 3 to avoid any bacterial growth. Solutions were filtered through 0.1 μm filters to remove any aggregated material, and then were split in two Eppendorf microcentrifuge tubes. Cef was dissolved in the same buffer and added to one tube for each pH to achieve a final Cef/HEWL molar ratio 2:1, in the other tube the same buffer volume was added. DLS measurements were taken during at each temperature interval between 20 and 90°C in 5°C increments, and a final measurement taken at 20°C post-thermal denaturation.

Computational methods
The computational methods used for data production are described in full detail in the accompanying article [1]. Briefly, ligand starting geometry and partial charges were obtained with the RESP procedure [9], using Ghemical 2.99.2 [10] and GAMESS [11] programs. Docking studies were performed with AutoDock 4.2 [12]. Amber16 package [13], with the ff14SB version of AMBER force field (FF) [14] for protein and, eventually, gaff FF [19] for the ligand was used for standard molecular dynamics (MD) simulations of Lysozyme-Cef complexes and constant pH-Replica Exchange MD (pH-REMD) of protein alone. Sequence alignments were obtained with UCSF Chimera program [15].