title: Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.
abstract: The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD.
authors: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.
authors: Jingjing
authors: J
authors: Li
authors: Izabela
authors: I
authors: Gierach
authors: Alison R
authors: AR
authors: Gillies
authors: Charles D
authors: CD
authors: Warden
authors: David W
authors: DW
authors: Wood
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D047668:Inteins / Q000235:genetics; D008024:Ligands; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
chemical_list: D008024:Ligands; D047495:PPAR gamma; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases