Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities.

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.