Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr)
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 occurre...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2024-07-01
|
| Series: | Biochemistry and Biophysics Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580824000554 |
| _version_ | 1797242974495571968 |
|---|---|
| author | Md. Forhad Hossain Shakhawat Hossain Md. Maisum Sarwar Jyoti Yuki Omori Saokat Ahamed Toshinobu Tokumoto |
| author_facet | Md. Forhad Hossain Shakhawat Hossain Md. Maisum Sarwar Jyoti Yuki Omori Saokat Ahamed Toshinobu Tokumoto |
| author_sort | Md. Forhad Hossain |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T18:47:45Z |
| format | Article |
| id | doaj.art-b4ca992092e242aaa29aa5b534fabcda |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-04-24T18:47:45Z |
| publishDate | 2024-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-b4ca992092e242aaa29aa5b534fabcda2024-03-27T04:52:22ZengElsevierBiochemistry and Biophysics Reports2405-58082024-07-0138101691Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr)Md. Forhad Hossain0Shakhawat Hossain1Md. Maisum Sarwar Jyoti2Yuki Omori3Saokat Ahamed4Toshinobu Tokumoto5Department of Bioscience, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, JapanBiological Science Course, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, JapanDepartment of Bioscience, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, JapanBiological Science Course, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, JapanDepartment of Bioscience, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, JapanDepartment of Bioscience, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Biological Science Course, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Corresponding author.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.http://www.sciencedirect.com/science/article/pii/S2405580824000554Graphene quantum dotsNuclear progesterone receptorProgesteroneFRETSteroids |
| spellingShingle | Md. Forhad Hossain Shakhawat Hossain Md. Maisum Sarwar Jyoti Yuki Omori Saokat Ahamed Toshinobu Tokumoto Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) Biochemistry and Biophysics Reports Graphene quantum dots Nuclear progesterone receptor Progesterone FRET Steroids |
| title | Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) |
| title_full | Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) |
| title_fullStr | Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) |
| title_full_unstemmed | Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) |
| title_short | Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr) |
| title_sort | establishment of a graphene quantum dot gqd based steroid binding assay for the nuclear progesterone receptor pgr |
| topic | Graphene quantum dots Nuclear progesterone receptor Progesterone FRET Steroids |
| url | http://www.sciencedirect.com/science/article/pii/S2405580824000554 |
| work_keys_str_mv | AT mdforhadhossain establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr AT shakhawathossain establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr AT mdmaisumsarwarjyoti establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr AT yukiomori establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr AT saokatahamed establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr AT toshinobutokumoto establishmentofagraphenequantumdotgqdbasedsteroidbindingassayforthenuclearprogesteronereceptorpgr |