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Outline • Introduction : computational method used • Paper discussion • Biotechnological applications Computational design of receptor and sensor proteins with novel functions Nature vol..423 May 8 2003, pp.:185-190 • Designed a computational method: – identify amino acid sequences predicted to form a complimentary surface between the protein and a target ligand replacing the wild type ligand Prot ein coordinat es 1 Poly-alanine PCS 2 Docking grid Ligand coordinat es 3 Rot at ional ligand ensemble Force field 4 Docked ligand ensemble 5 Ranked ligand ensemble Side-chain 6 rot amers Evolved PCS ensemble 7 Ranked PCS ensemble 8 Experiment s b • To test their method they chose E.coli. • Periplasmic binding protein (PBP): – – – – – glucose binding protein (GBP) ribose binding protein (RBP) arabinose binding protein (ABP) glutamine binding protein (QBP) histidine binding protein (HBP) • Target ligands bear little resemblance to wild type and chemically different from each other. • To explore critical parameters of molecular recognition: – molecular shape, chirality, functional groups, internal flexibility,charge, and water solubility. • How were they able to know if there is binding of the ligand ? • Introducing fluorophores at residues that exhibit changes in fluorescence emission • due to changes in conformation (open vs close) Test it in cells • RBP and GBP involved in signal transduction pathways at the gene levels • presence of ligand to the above mentioned receptors will cause the upregulation of b-galactosidase gene expression. • The RBP and GBP were replaced with the designed TNT and L-lactate receptors and tested using this expression mechanism . Biotechnological applications • Developing specific cell based detectors for chemical threats and pollutants: – TNT is carcinogen and explosive, having bacteria with TNT.R3 that respond with the expression of a fluorescent gene – Products such as drugs needs to be highly purified and with receptors being able to differentiate between chiral (L-lactate) compounds pharmaceutical companies will be able to achieve this goal