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.