Creagh A L, Koska J, Johnson P E, Tomme P, Joshi M D, McIntosh L P, Kilburn D G, Haynes C A
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Biochemistry. 1998 Mar 10;37(10):3529-37. doi: 10.1021/bi971983o.
Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.
差示扫描量热法已被用于研究纤维单胞菌β-1,4-葡聚糖酶CenC（CBDN1）的N端纤维素结合结构域的热稳定性和寡糖结合热力学。与其他小的单结构域球状蛋白相比，CBDN1具有相对较低的最大稳定性（在1℃和pH 6.1时，ΔGmax = 33 kJ/mol = 216 J/残基）。在pH 5.5至9之间，去折叠是完全可逆的，并且在pH 5.5至11之间符合两态平衡模型。当CBDN1中的单个二硫键被还原时，通过核磁共振光谱判断，该蛋白在所有条件下均保持去折叠状态。这表明分子内交联对CBDN1的稳定性有重要贡献。测得的去折叠热容量变化（ΔCp = 7.5 kJ mol-1 K-1）与根据去折叠时溶剂可及的非极性和极性表面积的预测变化计算得到的值非常吻合。将去折叠的比焓和熵外推至它们各自的收敛温度表明，对于孤立结构域CBDN1，每个残基的去折叠能量与Privalov（1）对许多单结构域球状蛋白所发现的能量一致。在存在各种浓度的纤维五糖的情况下，CBDN1去折叠的差示扫描量热图被拟合到一个描述蛋白质-配体结合与蛋白质去折叠之间联系的热力学模型。使用全局二维最小化程序对CBDN1-纤维五糖系统的结合焓、结合常数和去折叠热力学进行回归分析。外推得到的结合常数与在35℃下通过等温滴定量热法测定的值在数量上一致。
