Fernández A M, Villegas V, Martínez J C, Van Nuland N A, Conejero-Lara F, Avilés F X, Serrano L, Filimonov V V, Mateo P L
Departamento de Química Física, Universidad de Granada, Spain.
Eur J Biochem. 2000 Oct;267(19):5891-9. doi: 10.1046/j.1432-1327.2000.01638.x.
Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two alpha helices in order to increase their stability. At neutral and alkaline pH the three mutants, alpha-helix 1 (M1), alpha-helix 2 (M2) and alpha-helix 1 and alpha-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a beta conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third beta strands as well as part of alpha-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.
通过差示扫描量热法对人羧肽酶原A2的激活结构域ADA2h及其螺旋工程突变体进行了热力学表征。这些突变体是通过改变两个α螺旋暴露面上的残基构建的，以提高其稳定性。在中性和碱性pH条件下，三个突变体，即α螺旋1（M1）、α螺旋2（M2）以及α螺旋1和α螺旋2（DM），比野生型结构域更稳定，稳定性顺序为DM、M2、M1和野生型。在这些条件下，所有变体的圆二色光谱和核磁共振光谱非常相似，表明这种稳定性的增加并非总体结构变化的结果。量热分析表明，突变螺旋暴露于水表面的稳定作用似乎主要是熵效应，因为这些突变并未改变变性的焓或热容增加。在酸性条件下，所有变体的解折叠行为都会发生变化：野生型和M1有强烈的聚集倾向，在解折叠时形成β构象，而M2和DM则可逆解折叠，M2比DM更稳定。在pH 3.0条件下的圆二色光谱和核磁共振实验表明，涉及第二条和第三条β链残基以及部分α螺旋1的区域会改变其构象。似乎M2和DM改变构象后增强的稳定性降低了ADA2h在酸性pH下的聚集倾向。
