Thermal denaturation of the apo-cyclic AMP receptor protein and noncovalent interactions between its domains.
Cyclic AMP receptor protein (CRP) is allosterically activated by cAMP and functions as a global transcription regulator in enteric bacteria. Structural information on CRP in the absence of cAMP (apo-CRP) is essential to fully understand its allosteric behavior. In this study we demonstrated interdomain interactions in apo-CRP, using a comparative thermodynamic approach to the intact protein and its isolated domains, which were prepared either by limited proteolysis or using recombinant DNA. Thermal denaturation of the intact apo-CRP, monitored by differential scanning calorimetry, revealed an apparently single cooperative transition with a slight asymmetry. Combined with circular dichroism and fluorescence analysis, the thermal denaturation of apo-CRP could be interpreted as a coupled process involving two individual transitions, each attributable to a structural domain. When isolated individually, both of the domains exhibited significantly altered thermal behavior, thus pointing to the existence of non-covalent interdomain interactions in the intact apo-CRP. These observations suggest that the allosteric conformational change of CRP upon binding to cAMP is achieved by perturbing or modifying pre-existing interdomain interactions. They also underline the effectiveness of a comparative approach using calorimetric and structural probes for studying the thermodynamics of a protein.
Won
Hyung-Sik
HS
Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju 380-701, Korea. wonhs@kku.ac.kr
Seo
Min-Duk
MD
Ko
Hyun-Suk
HS
Choi
Wahn Soo
WS
Lee
Bong-Jin
BJ
D000494:Allosteric Regulation; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D001059:Apoproteins; D002373:Cyclic AMP Receptor Protein; D000242:Cyclic AMP