title: Engineering allostery.
abstract: Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. This approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Electronic address: sraman@genetics.med.harvard.edu.
authors.forename: Srivatsan
authors.initials: S
authors.lastname: Raman
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Taylor
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Genuth
authors.affiliation: Howard Hughes Medical Institute, Departments of Genome Sciences and Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors