20.500.12556/RUL-112849 Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR) This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. molecular biology computer simulation receptors adrenergic β2 – metabolism – chemistry peptidomimetics – chemical synthesis single-domain antibodies – chemistry true false true Angleški jezik Ni določen Članek v reviji 2019-11-15 10:10:32 2019-11-15 10:10:32 2022-08-25 03:51:37 0000-00-00 00:00:00 2019 0 0 13 str. art. 16555 Vol. 9 2019 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.2 2045-2322 10.1038/s41598-019-52934-8 4896890 18727432 RAZ_Sencanski_Milan_2019.pdf RAZ_Sencanski_Milan_2019.pdf 1 E1B7E895A81546ADF87578D94F6160AA 3594d525580ee3acc8efa4975960b2ba5e90150e4436c887fef3d09acb0e0ba9 b773541b-db2c-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144741 https://www.nature.com/articles/s41598-019-52934-8 Open access 1 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=124788 Biotehniška fakulteta Veterinarska fakulteta 0 0 0