Thermal denaturation of the apo-cyclic AMP receptor protein and noncovalent interactions between its domains.
Mol Cells
; 26(1): 61-6, 2008 Jul 31.
Article
em En
| MEDLINE
| ID: mdl-18525238
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.
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Bases de dados:
MEDLINE
Assunto principal:
Apoproteínas
/
Proteína Receptora de AMP Cíclico
/
Regulação Alostérica
Idioma:
En
Revista:
Mol Cells
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2008
Tipo de documento:
Article