Domain Coupling in a Multimodular Cellobiohydrolase CbhA From Clostridium Thermocellum

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Cbha, Cellobiohydrolase A, DSC Differential Scanning Calorimetry, GH Glycoside Hydrolase, CBD Carbohydrate-binding Domain, Ig Immunoglobulin-like Domain, X1 Domain of Unknown Function of Family 1 EDTA, Ethylenediaminetetraacetic Acid Multi-domain Protein, Domain Interactions, Protein Stability

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Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X11 and X12 domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X12, X11X12, CBD3, X11X12-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X11X12, and Ig-GH9-X11X12-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T d and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.

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FEBS Letters, v. 570, issue 20, p. 4367-4373