Quality control and biophysical characterisation data of VanSA

This data article presents the results from quality control experiments including N-terminal sequencing, SEC-MALS and Mass Spectrometry for purified VanSA used in experiments described in (Hughes et al., 2017) [1]; in addition to ligand interaction measurements and thermal melting curves of VanSA in the presence of screened ligands from circular dichroism measurements as well as UV–vis absorbance spectra for the binding interaction of VanSA in the presence of screened ligands.


a b s t r a c t
This data article presents the results from quality control experiments including N-terminal sequencing, SEC-MALS and Mass Spectrometry for purified VanS A used in experiments described in (Hughes et al., 2017) [1]; in addition to ligand interaction measurements and thermal melting curves of VanS A in the presence of screened ligands from circular dichroism measurements as well as UV-vis absorbance spectra for the binding interaction of VanS A  Amino acid sequencing for the recombinant expression of His 6 -tagged VanS A . Thermal melt profiles @ 225 nm for data summarised in Table 1 in Research article [1]. Binding interaction further monitored by UV-vis absorbance.

Data
Quality control measures for VanS A expression and purification included N-terminal sequencing, Mass Spectrometry and SEC-MALS. Protein of 41,510 7930 Da (Fig. 2) and N-terminal sequence MNSHM ( Fig. 1) was identified. 78% of the amino acid sequence for VanS A was identified by Mass Spectrometry to give a molecular weight of 45764 Da (Fig. 3). UV absorption measurements during CD titration experiments for VanS A as described in [1] (Fig. 4) were used for quality control of the experimentally determined dissociation constant (k d ). Change in CD at 225 nm was plotted against temperature and fitted with a Gibbs-Helmholtz equation for determination of melting temperature (T m ) (Fig. 5).

N-terminal sequencing
Purified proteins were separated by SDS-PAGE and electroblotted onto PVDF membrane before staining using Ponceau S for visualisation of the bands to be excised for sequencing by Edman degradation at Alta Bioscience Ltd, University of Birmingham, UK.

Size-exclusion chromatography multi-angle light scattering
Performed using 0.1 mg of purified protein in final suspension buffer. A Superdex 200 Increase 5/ 150 GL column (GE Healthcare Life Sciences) was pre-equilibrated with ddH 2 O overnight, followed by  ) was pre-equilibrated with 10 mM HEPES pH 8.0, 5% glycerol, 0.025% DDM before injection of 200 µl of VanS A (0.5 mg/ml) in 10 mM HEPES pH 8.0, 20% glycerol, 0.025% DDM using an ÄKTA pure system (GE Healthcare). Baseline set and the molecular weight calculated using data processing software (UNICORN 6, GE Healthcare). BSA as reference material was used to calibrate the instrument.  equilibration with running buffer containing 10 mM HEPES pH 8.0, 5% glycerol, 0.025% DDM before sample injection. A flow rate of 0.3 ml/min was used throughout.

Mass spectrometry
LC-MS/MS performed at Advanced Proteomics Facility, Department of Biochemistry, University of Oxford.
Peptides re-suspended in 10% formic acid were separated on an Ultimate 3000 UHPLC system (Thermo Fischer Scientific) and electrosprayed directly into a QExactive mass spectrometer (Thermo  Fischer Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Fischer Scientific). The peptides were trapped on a C18 PepMap100 pre-column (300 µm×5 mm,100 Å, Thermo Fisher Scientific) using solvent A (0.1% Formic Acid in water) at a pressure of 500 bar. Peptides were separated on a PepMapRSLC C18 column (2 um, 100 Å, 75 um×50 cm, Thermo Fisher Scientific) using a linear gradient (length: 120 minutes, 7% to 28% solvent B (0.1% formic acid in acetonitrile), flow rate: 200 nL/min). The raw data was acquired on the mass spectrometer in a data-dependent mode (DDA). Full scan MS spectra were acquired in the Orbitrap (scan range 350-2000 m/z, resolution 70000, AGC target 3e6, maximum injection time 50 ms). After the MS scans, the 20 most intense peaks were selected for HCD fragmentation at 30% of normalised collision energy. HCD spectra were also acquired in the Orbitrap (resolution 17500, AGC target 5e4, maximum injection time 120 ms) with first fixed mass at 180 m/z.
Raw MS data were processed by MaxQuant (version 1.5.0.35i) for peak detection and quantification. MS spectra were searched against a custom database using the Andromeda search engine with the following search parameters: full tryptic specifity, allowing two missed cleavage sites, fixed modification was set to carbamidomethyl (C) and the variable modification to acetylation (protein N-terminus), oxidation (M).
Mass spectra were recalibrated within MaxQuant with a precursor error tolerance of 50 ppm and then re-searched with a mass tolerance of 5 ppm.
Fragment ion tolerance was set to 20 ppm.

Synchrotron Radiation Circular Dichroism (SRCD), Circular Dichroism (CD) and UV-vis absorbance
SRCD spectroscopy was carried out in a nitrogen-flushed chamber at beamline B23 at the Diamond Light Source Ltd, Oxfordshire as described in [5,6]. For CD studies and UV-vis absorbance, experiments were conducted using a Chirascan-Plus (Applied Photophysics).
Ligand-containing samples were performed by addition of 5-fold molar equivalent of ligand stocks in 10 mM Tris. HCl pH 8.0 (control incubated with equivalent volume of 10 mM Tris-HCl pH 8.0). All samples were incubated at 20°C for 30 min prior to data collection. All data was analysed using CDApps [2] where the mean residue weight of VanS A was taken to be 113. Unless otherwise stated, all spectra presented are difference spectra where all relevant background buffers, ligands etc. have been subtracted. Data acquired when the HT of the detector (PMT) was equal to or greater than 600 V were excluded from the analyses. Far-UV measurements (180-260 nm) were commonly collected using 0.5 mg/ml of VanS A with bandwidth of 1 nm and 1 s integration time. Data presented in molar extinction (Δε). Temperature denaturation measurements were collected over the temperatures (20-95°C, in 5°C increments) in the absence and presence of ligands. Samples were incubated at the initial 20°C for 30 min after ligand or solvent addition prior to data acquisition. At each temperature step, reactions were incubated for 2 min prior to data collection (1 scan). A final scan was acquired post-temperature ramp after returning to 20°C and incubation for 20 minutes before data acquisition. Data was analysed using CDApps [1] to obtain difference spectra where all controls (buffers, ligands, etc.) had been subtracted. Change in CD (mdeg) at a specific wavelength was transferred to OriginPro® 9 and plotted against the corresponding temperature for fitting using Gibbs-Helmholtz equation derived from Boltzmann distribution [2,3] sigmoidal two-state denaturation curve to a Boltzmann distribution and the expression modified to include parameters for fitting of thermal denaturation data for the calculation of the melting temperature (T m ).
Near-UV measurements (260-350 nm) were collected using 1 mg/ml of protein using 10 mm pathlength cell, 2 nm bandwidth, 1 nm increments and 1 s integration. Titration experiments were conducted as described for standard near-UV measurements, with the modification of measurements collected after the addition of incremental volumes of ligand stock as described previously [7,8]. Change in CD (mdeg) a specific wavelength was monitored, the values transferred to OriginPro® and plotted against respective ligand concentration (M) and fitted with a hill1 binding [9] or biphasic dose response [10] function to determine the k d for binding.