{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["25(4)"],"submitter":["Frei CS"],"pubmed_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."],"journal":["Protein science : a publication of the Protein Society"],"pagination":["804-14"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC4941223"],"repository":["biostudies-literature"],"pubmed_title":["Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone."],"pmcid":["PMC4941223"],"pubmed_authors":["Sutter M","Qian S","Deutsch S","Frei CS","Wang Z","Cirino PC"],"additional_accession":[]},"is_claimable":false,"name":"Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone.","description":"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.","dates":{"release":"2016-01-01T00:00:00Z","publication":"2016 Apr","modification":"2020-11-19T08:12:52Z","creation":"2019-03-27T02:18:06Z"},"accession":"S-EPMC4941223","cross_references":{"pubmed":["26749125"],"doi":["10.1002/pro.2873"]}}