Hitrap desalt column

WT rLifA and rLifA^C1480A were exchanged into Assay Buffer (10 mM Tris, pH 7.6, 150 mM NaCl) using a HiTrap desalt column (GE Healthcare) at a flow rate of 4 mL/min, and centrifuged at 12,000g, 4 °C for 15 min to remove any dust particles that might interfere with size estimation. The mean hydrodynamic radius of a 1.1 µM solution of lymphostatin was measured using a Zetasizer Automated Plate Sampler (Malvern Instruments, UK) equipped with a 50 mW laser light source, with a wavelength of 830 nm. Data were collected at a scattering angle of 90° at 25 °C in 10 s accumulations repeated 13 times. The average autocorrelation data was fitted to a model optimised for protein solutions using the software supplied with the instrument. This analysis generated a distribution of particles by size.⁴⁹ (link) Experiments were repeated five times.

PDI proteins were reduced with 10 mM DTT for 10 min at room temperature. DTT was removed using a 5 ml of Hitrap desalt column (GE Healthcare) pre-equilibrated with 50 mM Tris/HCl, pH 7.5, buffer containing 150 mM NaCl and 1 mM EDTA (buffer A), according to the manufacturer's protocol. To oxidize the proteins, samples were incubated with 10 mM GSSG for 10 min at room temperature. GSSG was removed by gel filtration using a Superdex 10/300 GL column (GE Healthcare) pre-equilibrated with buffer A. The redox status of all PDI proteins was verified by carrying out AMS alkylation and separation by SDS/PAGE.

The far ultraviolet (UV) CD spectra of 0.11 µM WT rLifA and rLifA^C1480A were recorded at 10 nm/min; data pitch 0.1 nm; response time 2 s between 185 and 285 nm in a 0.1 cm path length quartz cuvette at 25 °C (Jasco-810 spectropolarimeter). The proteins were exchanged into 10 mM NaH₂PO₄, pH 7.6, 100 mM NaF prior to analysis using a HiTrap desalt column (GE Healthcare) at a flow rate of 4 mL/min. Spectra were corrected by subtracting a buffer baseline, each an average of five spectra, acquired under the same conditions. Secondary structure was estimated using the DichroWeb CD secondary structure analysis server⁵² (link) including the methods CONTIN, SELCON3 and CDSSTR53 (link), 54 (link), 55 (link), 56 (link) and reference data sets 3, 4, 6, 7, SP175 and SMP180.

Isothermal titration calorimetry (ITC) experiments were carried out to determine the affinity of Cyp40 for Hsp90 peptide over the temperature range 10°C–30°C at 5°C intervals (Auto-iTC200 microcalorimeter; Malvern Instruments). Cyp40 was exchanged into the experimental buffer; 50 mM Hepes, pH 8; 100 mM sodium chloride; 1 mM DTT (HiTrap desalt column, GE Healthcare Life Sciences) immediately before the experiment. SRMEEVD (500 μM) was titrated into 7.7 μM Cyp40 in 15 × 2.5 μl aliquots. Data were analysed using non-linear regression in the MicroCal ORIGIN software package assuming a 1:1 binding model.

The temperature-induced unfolding of lymphostatin was followed using the environmentally sensitive dye SYPRO Orange (Invitrogen). SYPRO Orange shows an increase in fluorescence correlated with the exposure of hydrophobic residues upon unfolding.44 (link), 45 (link) The fluorescence of WT rLifA and rLifA^C1480A (both 0.1 µM) in 5 × SYPRO Orange was measured between 15 and 70 °C at 0.5 °C increments every 30 s (Bio-Rad IQ5 Multicolor Real-Time PCR Detection System). The proteins were exchanged into Assay Buffer prior to analysis using a HiTrap desalt column (GE Healthcare) at a flow rate of 4 mL/min. Relative fluorescence units (RFU) for each sample were plotted against temperature and the unfolding transition temperature (T_m) of each protein was calculated from the steepest part of the melting curve. Experiments were repeated in triplicate.