Study from this laboratory shows that Escherichia coli cAMP receptor protein (CRP) exist in at least three conformational states - CRP, CRP-cAMP and CRP-(cAMP)2. To relate this structural information to the functional state of CRP, it is proposed to determine: (A) The nature of conformational changes involved in CRP activation and inactivation. Since the three conformational states of CRP, detected in this laboratory, apparently exhibit different affinities towards cAMP and DNA, a detail characterization of these states by a unique combination of biophysical techniques is proposed. The global structural changes induced by ligands will be monitored by analytical gel chromatography. X-ray scattering will be employed in conjunction with molecular modelling based on crystallographic data to define the conformational changes as viewed from changes in domain interactions. To monitor the change in dynamic motions in CRP tritium exchange will be employed. Further development of this procedure could lead to identification of peptides responsive to allosteric changes induced by cAMP, RNA polymerase or DNA binding. The presence of segmental flexibility in between the DNA and cAMP binding domains and the potential importance of this dynamic motion on CRP function can be assessed by fluorescence depolarization. The pathways of message transmission will be probed by studies on hybrids of CRP formed from modified CRP generated through site directed mutagenesis. (B) The functional properties of these conformational states of CRP. Since CRP and the CRP-cAMP complex have been shown to have low and high affinities for specific DNA sites, respectively, what is the physiological significance of CRP-(cAMP)2 complex? The affinity of the second binding site for cAMP is very weak, so does it have any role in the mechanism of control? Since the complete system of transcription involves interactions among cAMP, CRP, RNA polymerase, specific and nonspecific DNA sites, the linked thermodynamics involved in the interactions among these components will be monitored. These measurements represent the first quantitative attempt to describe the thermodynamics of the complete system.
