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Elucidating residue roles in engineered variants of AraC regulatory protein

2009; Wiley; Volume: 19; Issue: 2 Linguagem: Inglês

10.1002/pro.310

1469-896X

Shuang‐Yan Tang, Patrick C. Cirino,

Bacterial Genetics and Biotechnology

The AraC regulatory protein was previously engineered to control gene expression specifically in response to D-arabinose and not the native effector L-arabinose (Tang et al., J Am Chem Soc 2008;130:5267-5271). Mutations were targeted in the ligand-binding pocket and on the AraC N-terminal arm, which plays an important role in maintaining repressing or activating conformations in the absence or presence of effector, respectively. In this study, we analyze the contributions of individual mutations toward the overall mutant functions in an attempt to streamline future AraC design efforts. For a variety of point mutants, we quantify the induced expression response to D-arabinose (level of leaky expression, induction fold, half-maximal dose response, and effector specificity) and the binding affinity of the purified ligand-binding domain for D-arabinose. We find that mutations introduced in the N-terminal arm (design Position 8) strengthen the induction response, most likely by weakening interactions with the DNA-binding domain, but are not involved in ligand binding. Meanwhile, binding pocket mutations occurring further away from the arm (Positions 80 and 82) primarily contribute to maintaining repression in the absence of effector and do not show response to D-arabinose without the accompanying mutations. Combinations of mutations cooperatively couple molecular recognition to transcriptional activation, demonstrating the complexity of the AraC regulatory switch and the power of combinatorial protein design to alter effector specificity while maintaining regulatory function.