Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr)

Abstract

Previously, we established a homogeneous assay for membrane progesterone receptor alpha (mPRα) ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to mPRα. When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC), fluorescence occurred by fluorescence resonance energy transfer (FRET) but was reduced by the ligand-receptor binding activity. The established way showed ligand specificity as mPRα protein. In this study, we tried to establish the same way for nuclear progesterone receptor (Pgr). The ligand-binding domain (LBD) of zebrafish Pgr (zPgrLBD) was expressed as a fusion protein with glutathione S-transferase (GST) (GST-zPgrLBD). The recombinant protein was then purified and coupled with GQDs to produce GQD-conjugated GST-zPgrLBD (GQD-GST-zPgrLBD). When mixed with a P4-BSA-FITC and activated by 370 nm light, fluorescence at 520 nm appeared by FRET mechanism. Fluorescence at 520 nm was reduced by adding free progesterone to the reaction mixture. Reduction of fluorescence was induced by zPgr ligands but not by steroids or chemicals that do not interact with zPgr. The results showed the formation of a complex of GQD-GST-zPgrLBD and P4-BSA-FITC with ligand-receptor binding. The binding of the compounds was further confirmed by a radiolabeled steroid binding assay. A homogenous ligand-binding assay for nuclear progesterone receptor has been established.

Keywords: FRET; Graphene quantum dots; Nuclear progesterone receptor; Progesterone; Steroids.

Fig. 1

Expression and purification of the ligand-binding domain (LBD) of zebrafish Pgr as a fusion protein with GST and production of GQD-coupled GST-zPgrLBD. A. A diagram of the produced recombinant GST-fusion zPgrLBD. The predicted ligand-binding domain at the C-terminus is indicated by black boxes. The GST tag at the N-terminus is indicated by a white box. B. SDS-PAGE analysis of purified recombinant GST-zPgrLBD. Protein bands were detected by CBBR staining (CBBR) or immunostained with anti-GST-tag antibody (α-GST). C. Western blot analysis of GST-zPgrLBD and GQD-GST-zPgrLBD. The protein band of GST-zPgrLBD is indicated by an arrowhead. The bands of GQD-GST-zPgrLBD are indicated by a parenthesis.

Fig. 2

Fluorescence characteristics of GQD-GST-zPgrLBD and competition of binding of P4-BSA-FITC with GQD-GST-zPgrLBD by steroids and their analogues. (A) The fluorescent scanning pattern of free GQD-GST-zPgrLBD is indicated in blue line. Fluorescent scanning pattern of the reaction mixture with (orange line) or without free P4 (green line). The difference of fluorescence at 520 nm caused by the addition of free P4 is indicated by the double-sided arrow. (B) The dose-dependent effects of steroids (progesterone (P4), 17β-estradiol (E2), testosterone (T), cortisol) and their analogues (17,20β-DHP and Org OD 02) were determined using the established assay. An assay was performed in triplicate for each compound. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Competition by steroids and progestins for binding to GQD-GST-zPgrLBD. Samples were incubated with 2 nM [³H]-17,20β-DHP and 1 μM of competitor. An assay was performed in triplicate for each competitor. The average of the three assays is indicated with standard deviation. Competition for [³H]-17,20β-DHP binding are expressed as a percentage of maximum specific progesterone (P4) binding. The competitors analyzed were progesterone (P4); 17,20β-DHP; Org OD 02; 17β-estradiol (E2); testosterone (T); Cortisol.