title: Monitoring DNA binding to Escherichia coli lactose repressor using quartz crystal microbalance with dissipation.
abstract: Lactose repressor protein (LacI) functions as a negative transcription regulator in Escherichia coli by binding to the operator DNA sequence. Our understanding of the immobilized LacI function and the effect of ligand binding on the conformation of LacI-DNA complexes remains poorly understood. Here, we have examined the difference in functionality of wild-type and mutant LacI binding to the target DNA using quartz crystal microbalance with dissipation (QCM-D). To direct the orientation of LacI binding to the gold surface, residue 334 was substituted with cysteine (T334C) to generate a sulfur-gold linkage. Position 334 is located on the surface opposite the DNA-binding domain and remote from the site for inducer binding. With T334C immobilized on the gold surface, our sensors successfully detect operator binding as well as the release of the operator DNA from the repressor in the presence of inducer isopropyl-β-D-thiogalactoside (IPTG). Besides the natural operator DNA sequence (O(1)), a symmetric high-affinity DNA sequence (O(sym)), and a non-specific DNA (O(ns)) sequence with low affinity were also used. In addition, the impact of anti-inducer o-nitrophenyl-beta-d-fucoside (ONPF), which stabilizes LacI operator binding, was examined. The results from immobilized mutant LacI are in good agreement with known solution parameters for LacI-ligand interactions, demonstrating that QCM-D provides a rapid and efficient measurement of DNA binding and impact of ligands upon binding for this complex oligomeric protein.
authors.affiliation: Department of Biochemistry and Cell Biochemistry, Rice University, Houston, Texas 77005, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Xu
authors.forename: Kai-Wei
authors.initials: KW
authors.lastname: Liu
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Matthews
authors.forename: Sibani L
authors.initials: SL
authors.lastname: Biswal
mesh_terms: D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D055767:Immobilized Proteins / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism*; D011485:Protein Binding; D058525:Quartz Crystal Microbalance Techniques
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D056968:Lac Repressors