Calcium and Domain Interactions Contribute to The Thermostability of Domains of The Multimodular Cellobiohydrolase, Cbha, a Subunit of The Clostridium Thermocellum Cellulosome

Authors

Irina Kataeva, University of Georgia
Vladimir N. Uversky, University of California, Santa CruzFollow
Lars G. Ljungdahl, University of Georgia

Document Type

Article

Publication Date

2003

Keywords

Domain-structured Polypeptide, Glycoside Hydrolase, Multimodular Protein, Stabilization of Protein Fold, Temperature-induced Denaturation, Thermodynamics

Digital Object Identifier (DOI)

https://doi.org/10.1042/bj20021621

Abstract

Each of three internal domains of multi-modular cellobiohydrolase CbhA from Clostridium thermocellum, X1₁, X1₂ (previously designated as fibronectin type 3-like modules, Fn3₁ and Fn3₂) and family 3 carbohydrate-binding module (CBM3) binds 1 mol of Ca²⁺. Structures and thermal stabilities of X1₁, X1₂, CBM3, X1₁X1₂, and X1₁X1₂-CBM3 containing Ca²⁺ (holo-proteins) and without Ca²⁺ (apo-proteins) have been studied using CD spectroscopy. All domains are β-proteins with irregular far-UV CD spectra due to the aromatic side chain contributions. The positive signal at 294 nm in the near-UV CD spectrum of X1₁ lacking a tryptophan residue might be attributed to the presence of aromatic clusters. Thermal denaturation of all proteins is reversible and results in the total loss of tertiary structure and preservation of significant amount of ordered secondary structure. Removal of Ca²⁺ destabilizes polypeptides in a different way and to a different extent. It decreases the melting temperature (T_m) (by 20 °C) and co-operativity of thermal transition of X1₁, increases the number of transitions and lowers the co-operativity of unfolding of CBM3, and slightly decreases T_ms (2.4–4.2 °C) of X1₂, X1₁X1₂, and X1₁X1₂-CBM3. Transitions of X1₁X1₂ and X1₁X1₂-CBM3 follow a two-state model regardless of the presence of Ca²⁺. X1₁ is strongly stabilized in the apo-X1₁X1₂ and apo-X1₁X1₂-CBM3 as they display T_ms similar to those of individual and combined holo-modules. Observed CD spectra of X1₁X1₂ and X1₁X1₂-CBM3 differ from those calculated as the simple weighted sum of individual modules. These differences are more prominent in spectra of apo-proteins. The results indicate the presence of inter-domain interactions in CbhA. Holo-modules, i.e. containing Ca²⁺, behave essentially independently, but in the absence of Ca²⁺ domain interactions are more important for the conformation of the polypeptides.

Was this content written or created while at USF?

Yes

Citation / Publisher Attribution

Biochemistry, v. 372, issue 1, p. 151-161

Scholar Commons Citation

Kataeva, Irina; Uversky, Vladimir N.; and Ljungdahl, Lars G., "Calcium and Domain Interactions Contribute to The Thermostability of Domains of The Multimodular Cellobiohydrolase, Cbha, a Subunit of The Clostridium Thermocellum Cellulosome" (2003). Molecular Medicine Faculty Publications. 707.
https://digitalcommons.usf.edu/mme_facpub/707