Thermal denaturation of the apo-cyclic AMP receptor protein and noncovalent interactions between its domains

• Published in: Molecules and cells Vol. 26; no. 1; pp. 61 - 66
• Main Authors: Won, H.S. (Konkuk University, Chungju, Republic of Korea), E-mail: wonhs@kku.ac.kr, Seo, M.D. (Seoul National University, Seoul, Republic of Korea), Ko, H.S. (Konkuk University, Chungju, Republic of Korea), Choi, W.S. (Konkuk University, Chungju, Republic of Korea), Lee, B.J. (Seoul National University, Seoul, Republic of Korea), E-mail: lbj@nmr.snu.ac.kr
• Format: Journal Article
• Language: English
• Published: United States 한국분자세포생물학회 31.07.2008
• Subjects: ADN; Allosteric Regulation; allostery; Apoproteins - chemistry; Apoproteins - metabolism; Calorimetry, Differential Scanning; Circular Dichroism; Cyclic AMP - metabolism; cyclic AMP receptor protein; Cyclic AMP Receptor Protein - chemistry; Cyclic AMP Receptor Protein - metabolism; differential scanning calorimetry; DNA; Escherichia coli - genetics; Escherichia coli - metabolism; fluorescence spectroscopy; interdomain interaction; Protein Binding; Protein Denaturation; Protein Structure, Tertiary; thermal denaturation; Thermodynamics; 생물학
• ISSN: 1016-8478; 0219-1032
• DOI: 10.1016/S1016-8478(23)13964-1

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.