title: Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone.
abstract: The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to l-arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named "AraC-TAL1") was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand-binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence-activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X-ray diffraction was used to solve the crystal structure of the apo AraC-TAL1 ligand-binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild-type AraC ligand-binding domain (root-mean-square deviation 0.93 Å), suggesting that AraC-TAL1 behaves similar to wild-type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence-function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, 77204.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Frei
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, 77204.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, 77204.
authors.forename: Shuai
authors.initials: S
authors.lastname: Qian
authors.affiliation: Joint Genome Institute, 2800 Mitchell Drive Walnut Creek, California, 94598.
authors.forename: Samuel
authors.initials: S
authors.lastname: Deutsch
authors.affiliation: Joint Genome Institute, 2800 Mitchell Drive Walnut Creek, California, 94598.
authors.forename: Markus
authors.initials: M
authors.lastname: Sutter
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, 77204.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Cirino
mesh_terms: D019943:Amino Acid Substitution; D051777:AraC Transcription Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011753:Pyrones / Q000378:metabolism*; D011897:Random Allocation
chemical_list: D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D011753:Pyrones; C030720:triacetic acid lactone
keywords: AraC; FACS; biosensor; cooperative residues; crystal structure; directed evolution; high-throughput screening; molecular reporter; regulatory protein