CRP is a homodimeric transcription factor that regulates gene expression in bacteria as a function of cAMP concentration. Each CRP monomer has a cAMP-binding and a DNA-binding domain. Binding of cAMP to the homodimer allosterically controls CRP activity by causing a conformational change in the protein that promotes DNA binding.
Our goal is to dissect the linkage between cAMP binding, DNA recognition and conformational changes in CRP. To do so, we engineered a single-chain CRP dimer in which both CRP monomers are fused by a flexible polypeptide linker. Using this novel construct, we place asymmetric mutations that perturb DNA binding or cAMP binding in only one subunit, thereby allowing us to evaluate the propagation of mutational effects across the dimer interface. In conjunction with our re-engineered single chain CRP dimer, we use isothermal titration calorimetry, fluorescence spectroscopy, circular dichroism and optical tweezers to establish a relationship between the allosteric behavior of the protein (i.e. DNA binding affinity and cAMP binding cooperativity) and fine-tuning of stability and dynamics.
Gárate F, Dokas S, Fe Lanfranco M, Canavan C, Wang I, Correia JJ, Maillard RA. (2021) cAMP is an allosteric modulator of DNA binding specificity in cAMP receptor protein from Mycobacterium tuberculosis. J Biol Chem. 296: 100480.
Lanfranco MF, Gárate F, Engdahl AJ, Maillard RA. (2017) Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery. J Biol Chem. 292 (15): 6086-6093
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