Site-selective covalent immobilization of PPARγ using a label-free strategy for chromatographic study

Abstract

The development of site-selective covalent protein immobilization strategies is crucial for many protein-based bioanalytical assays. Herein, we proposed a label-free, site-selective, covalent immobilization strategy for the functionalization of PPARγ on GW9662 modified macroporous silica gels. The gels were characterized by X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). Molecular docking in combination with competitive study, adsorption energy distribution (AED) calculation, and frontal analysis were used to investigate the binding of ligands with GW9662 occupied PPARγ. Docking results indicated that rosiglitazone, pioglitazone, and gensenoside Rh1 could cooperatively bind to the receptor with GW9662. Competitive studies revealed that the three ligands bond with PPARγ through the same site in the presence of GW9662. AED calculation showed one type of binding site for the three ligands on the receptor. Due to the presence of GW9662, the affinities of the three ligands ((2.35 ± 0.05) × 105 M−1 for rosiglitazone, (1.27 ± 0.02) × 105 M−1 for pioglitazone, and (3.18 ± 0.13) × 105 M−1 for gensenoside Rh1) with PPARγ were one or two orders of magnitude smaller than the literature reported values. The good performance of the immobilized PPARγ provided an opportunity for application in receptor-ligand interactions and cooperative partner screening. Our strategy is, therefore, a promising way to realize the label-free, covalent immobilization of functional proteins.