| Autor: |
Kögler LM; Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, Leipzig, Germany., Stichel J; Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, Leipzig, Germany., Kaiser A; Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, Leipzig, Germany., Beck-Sickinger AG; Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, Leipzig, Germany. |
| Jazyk: |
angličtina |
| Zdroj: |
Frontiers in pharmacology [Front Pharmacol] 2019 Mar 01; Vol. 10, pp. 176. Date of Electronic Publication: 2019 Mar 01 (Print Publication: 2019). |
| DOI: |
10.3389/fphar.2019.00176 |
| Abstrakt: |
G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y 2 receptor (Y 2 R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y 2 R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y 2 R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p -benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y 1 R, NPY position 27 remains flexible when bound to Y 2 R. These results are in agreement with the suggested binding mode of NPY at Y 2 R. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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