Stability and Oligosaccharide Binding of the N1 Cellulose-Binding Domain of Cellulomonas fimi Endoglucanase CenC

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• 作者：A. Louise Creagh ; Jü ; rgen Koska ; Philip E. Johnson ; Peter Tomme ; Manish D. Joshi ; Lawrence P. McIntosh ; Douglas G. Kilburn ; and Charles A. Haynes
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：March 10, 1998
• 年：1998
• 卷：37
• 期：10
• 页码：3529 - 3537
• 全文大小：124K
• 年卷期：v.37,no.10(March 10, 1998)
• ISSN：1520-4995

文摘

Differential scanning calorimetry has been used to study thethermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-bindingdomain of Cellulomonas fimi es/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-1,4-glucanase CenC (CBD_N1). CBD_N1 has arelatively low maximum stability (es/gifchars/Delta.gif" BORDER=0 >G_max = 33kJ/mol = 216J/residue at 1 es/entities/deg.gif">C and pH 6.1) compared to other small single-domainglobular proteins. The unfoldingis fully reversible between pH 5.5 and 9 and in accordance with thetwo-state equilibrium model betweenpH 5.5 and 11. When the single disulfide bond in CBD_N1is reduced, the protein remains unfolded at allconditions, as judged by NMR spectroscopy. This indicates that theintramolecular cross-link makes amajor contribution to the stability of CBD_N1. Themeasured heat capacity change of unfolding(es/gifchars/Delta.gif" BORDER=0 >C_p =7.5 kJ mol^-1 K^-1)agrees well with that calculated from the predicted changes in thesolvent accessiblenonpolar and polar surface areas upon unfolding. Extrapolation ofthe specific enthalpy and entropy ofunfolding to their respective convergence temperature indicates thatper residue unfolding energies forCBD_N1, an isolated domain, are in accordance with thosefound by Privalov (1) for many single-domainglobular proteins. DSC thermograms of the unfolding ofCBD_N1 in the presence of variousconcentrationsof cellopentaose were fit to a thermodynamic model describing thelinkage between protein-ligand bindingand protein unfolding. A global two-dimensional minimizationroutine is used to regress the bindingenthalpy, binding constant, and unfolding thermodynamics for theCBD_N1-cellopentaose system.Extrapolated binding constants are in quantitative agreement withthose determined by isothermal titrationcalorimetry at 35 es/entities/deg.gif">C.