Azuaga A I, Conejero-Lara F, Rivas G, De Filippis V, Fontana A, Mateo P L
Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Spain.
Biochim Biophys Acta. 1995 Sep 27;1252(1):95-102. doi: 10.1016/0167-4838(95)00129-i.
The 205-316 C-terminal fragment of thermolysin has been studied by differential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimental conditions. The effect of sample concentration at the different pH values on the calorimetric traces is consistent with a monomer-dimer equilibrium of the folded fragment, which undergoes thermal unfolding into individual fragments. Equilibrium sedimentation experiments at 10 degrees C and different pH values confirm the presence of the association equilibrium and provide the value of the dimerization constants. The global analysis of the calorimetric, heat capacity curves has been carried out by a multidimensional fitting to the model N2<-->2N<-->2U. The analysis leads to a complete thermodynamic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a partially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information currently available and compared with the energetics of unfolding of the shorter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lara, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases the relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment.
已通过差示扫描量热法在pH值为2.5、3.0、3.5、4.0和5.0以及恒定离子强度为130 mM的条件下研究了嗜热菌蛋白酶的205 - 316 C末端片段。在我们的实验条件下，该片段的热解折叠发生在热力学平衡状态。不同pH值下样品浓度对量热曲线的影响与折叠片段的单体 - 二聚体平衡一致，该平衡片段会热解折叠成单个片段。在10℃和不同pH值下进行的平衡沉降实验证实了缔合平衡的存在，并提供了二聚化常数的值。通过对模型N2<-->2N<-->2U进行多维拟合，对量热、热容曲线进行了整体分析。该分析得出了片段缔合和解折叠过程的完整热力学特征。所得的热力学函数表明单体和二聚体片段均具有部分解折叠结构，以及与缔合过程相关的构象变化。我们根据目前可用的结构信息对结果进行了讨论，并与嗜热菌蛋白酶较短的255 - 316二聚体C末端片段（Conejero-Lara, F., De Filippis, V., Fontana, A.和Mateo, P.L. (1994) FEBS Lett. 344, 154 - 156）的解折叠能量进行了比较。额外50个残基的存在增加了205 - 316单体片段相对于255 - 316片段的相对丰度。
