title: Engineering alternate cooperative-communications in the lactose repressor protein scaffold.
abstract: To expand our understanding of the hallmarks of allosteric control we used directed-evolution to engineer alternate cooperative communication in the lactose repressor protein (LacI) scaffold. Starting with an I(s) type LacI mutant D88A (i.e. a LacI variant that is insensitive to the exogenous ligand isopropyl-β-d-thiogalactoside (IPTG) and remains bound to operator DNA, + or -IPTG) we used error-prone polymerase chain reaction to introduce compensatory mutations to restore modulated DNA binding function to the allosterically 'dead' I(s)(D88A) background. Five variants were generated, three variants (C4, C32 and C80) with wild-type like function and two co-repressor variants (C101 and C140) that are functionally inverted. To better resolve the residues that define new allosteric networks in the LacI variants, we conducted mutational tolerance analysis via saturation mutagenesis at each of the evolved positions to assess sensitivity to mutation--a hallmark of allosteric residues. To better understand the physicochemical bases of alternate allosteric function, variant LacI(C80) was characterized to assess IPTG ligand binding at equilibrium, kinetically using stopped-flow, and via isothermal titration calorimetry. These data suggest that the conferred modulated DNA binding function observed for LacI(C80), while thermodynamically similar to wild-type LacI, is mechanistically different from the wild-type repressor, suggesting a new allosteric network and communication route.
authors.affiliation: Department of Chemical & Environmental Engineering, Yale University, 55 Prospect Street, Malone Engineering Center 214, New Haven, CT, USA.
authors.forename: Sarai
authors.initials: S
authors.lastname: Meyer
authors.forename: Roee
authors.initials: R
authors.lastname: Ramot
authors.forename: Krishna
authors.initials: K
authors.lastname: Kishore Inampudi
authors.forename: Beibei
authors.initials: B
authors.lastname: Luo
authors.forename: Chenyu
authors.initials: C
authors.lastname: Lin
authors.forename: Swathi
authors.initials: S
authors.lastname: Amere
authors.forename: Corey J
authors.initials: CJ
authors.lastname: Wilson
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D007700:Kinetics; D056968:Lac Repressors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D056968:Lac Repressors; D008024:Ligands; D007544:Isopropyl Thiogalactoside
keywords: allostery; lactose repressor; protein engineering