9722884 Schwarz In E. coli. the transcription of over 20 genes for enzymes involved in sugar metabolism is activated by cAMP receptor protein (CRP) which has undergone a conformational change induced by the binding of cyclic adenosine monophosphate (cAMP) to CRP. Amino-acid substitutions in CRP, however, dramatically alter the level of its transcriptional activity. To elucidate how mutations alter the level of transcriptional activation and ultimately the mechanism for transcriptional activation, the research will consist of (1) determining the thermodynamic linkages between the binding reactions of CRP/mutants with cNMP, DNA, and RNA polymerase from calorimetry measurements and (2) determining the structures of the cNMP-ligated complexes from x-ray crystallography measurements. Small angle neutron scattering measurements, differential scanning calorimetry, and protease probes will be employed to substantiate these structural differences in solution. The level of transcription by the cNMP-ligated CRP/mutant complexes will be quantitatively determined by a well-defined in vitro assay. Correlations between the conformational changes and the thermodynamic linkages will ascertain how conformational changes control the thermodynamics of transcription. Comparison of the levels of in vitro and in vivo transcriptional activation may lead to the discovery of secondary events which also influence the level of transcriptional activation in the cell. Many biological processes are initiated by conformational changes in proteins induced by specific protein-ligand interactions. The goal of this research is to provide a better understanding of how ligand binding at sites far removed from the active site of a protein control its level of activity through conformational changes induced by binding of the ligand. In addition, it will elucidate how small structural alterations in the DNA binding site of proteins control the high specificity of DNA-binding proteins which exhibit similar topologies at the D NA binding site, e.g. the eukaryotic homolog of CRP, the cAMP-dependent protein kinase. Ultimately, this knowledge will lead to a better understanding of the relationship between structure and function in biological systems. ***
