Domain coupling in a multimodular cellobiohydrolase CbhA from Clostridium thermocellum.
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), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-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.
Kataeva
Irina A
IA
Department of Biochemistry and Molecular Biology, A216 Fred Davison Life Sciences Complex, University of Georgia, Athens, GA 30602, USA. kataeva@uga.edu
Brewer
John M
JM
Uversky
Vladimir N
VN
Ljungdahl
Lars G
LG
D002118:Calcium / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry*; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D043366:Cellulose 1,4-beta-Cellobiosidase; D002118:Calcium