Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Thermal Denaturation of the Apo-cyclic AMP Receptor Protein and Noncovalent Interactions between Its Domains

  • Won, Hyung-Sik (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Seo, Min-Duk (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University) ;
  • Ko, Hyun-Suk (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Choi, Wahn Soo (Department of Immunology, College of Medicine, Konkuk University) ;
  • Lee, Bong-Jin (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University)
  • Received : 2007.11.16
  • Accepted : 2008.02.05
  • Published : 2008.07.31
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Abstract

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.

Keywords

Acknowledgement

Supported by : Korea Research Foundation

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