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 (FACS). Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine total 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 A), suggesting 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. This article is protected by copyright. All rights reserved.