Abstract
We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his‐tagged human cyclophilin‐A. Our orientation‐specific stabilisation approach captures his‐tagged protein under ‘physiological conditions’ (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni2+‐nitrilotriacetic acid surfaces, very briefly activated for primary amine‐coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin‐A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo‐kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin‐A–cyclosporin‐A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin‐A, from a screen of a fragment library. This fragment, 2,3‐diaminopyridine, bound specifically with a mean affinity of 248 ± 60 μm. The X‐ray structure of this 109‐Da fragment bound in the active site of cyclophilin‐A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin‐A inhibitors.
Highlights
We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A
Cell pellets were resuspended in 20 mM NaH2PO4, pH 7.4; 500 mM NaCl; 20 mM imidazole; plus protease inhibitors at 10% w/v, and lysed at 6 °C by a single passage through a Constant Systems Cell Disruptor (1.1 kW TS Benchtop) set at 22 kpsi, followed by centrifugation at 50 000 g for 1 h at 4 °C
We have streamlined a protocol for His-CypA purification and Fig. 1 shows the typical high purity (≥ 95%), monodispersity and activity of the protein used in all our experiments
Summary
This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilinA–cyclosporin-A interaction. A threefold concentration series of CsA ranging from 2.47 to 200 nM, in 10 mM NaH2PO4, pH 7.5, 150 mM NaCl, 1 mM EDTA; 0.05% surfactant P20; 1% ethanol, was injected over the sensor surface, at 100 lLÁminÀ1 with 60 s contact and dissociation times.
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