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Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For <i>E. coli lac</i> repressor (LacI)<i>, </i>the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both <i>in vitro</i> on DNA microarrays with 2,479 different Lac operators and<i> in vivo </i>via single-molecule experiments. We identified a mutation that enhances the specificity but reduces binding strength globally, and another mutation that makes the operator binding stronger but also reduces the specificity. However, the altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity–inducibility trade-offs in LacI, our data support the model in which the trade-off is between binding stability and inducibility rather than between speed and binding stability.This repository contains the molecular dynamics (MD) simulation datasets, analysis scripts, and code necessary to reproduce the results of our study. It also provides information on the datasets from single-molecule live-cell experiments and Protein Binding Microarray (PBM) assays, which are available in the BioImage Archive.File list:Analysis: Analysis.tar - For plotting all figures in the main text and supplementaryCode: Code.tar - Utility scriptsData:Folders with prefix: 'Microscopy_Data_' - DataSets for Protein Binding Microarray experiments and Single Molecule experiments (raw fluorescence images), download these data from the link provided in 'Related material'.Folders with prefix: 'MD_Data' - Data set for molecular dynamics simulations.README.txt: Instructions for setting up folder's structure and coding environment for running this project.<br>