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KMID : 0578320080260010061
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Molecules and Cells
2008 Volume.26 No. 1 p.61 ~ p.66
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Thermal Denaturation of the Apo-cyclic AMP Receptor Protein and Noncovalent Interactions between Its Domains
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Won Hyung-Sik
Lee Bong-Jin
Choi Wahn-Soo
Seo Min-Duk
Ko Hyun-Suk
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Abstract
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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.
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KEYWORD
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allostery, circular dichroism, cyclic AMP receptor protein, differential scanning calorimetry, fluorescence spectroscopy, interdomain interaction, thermal denaturation
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