An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

Abstract

The binding of cyclic (c)AMP and cGMP to the cyclic receptor protein (CRP) from E. coli was investigated by equilibrium dialysis at pH 8.0 and 20.degree. C at different ionic strengths (0.05-0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for 2 identical sites and site/site interactions with the interaction free energy-RT ln .alpha., and the intrinsic binding constant K and cooperativity parameter .alpha. were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and .alpha. are not very different from that for cAMP. Since 2 binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded (ds)DNA, CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter .alpha. was very sensitive to changes of pH between 6.0 and 8.0, at 0.2 .MU. ionic strength and 20.degree. C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are .DELTA.H.degree. = 1.7 kcal.cntdot.mol-1 and .DELTA.S.degree. = 15.6 entropy units, respectively.