Hiraga K, Yutani K
Institute for Protein Research, Osaka University, Japan.
Protein Eng. 1996 May;9(5):425-31. doi: 10.1093/protein/9.5.425.
To eludicate the role in conformational stability of Cys residues buried in the interior of a protein, the thermodynamic properties of denaturation of mutant alpha subunit of Escherichia coli tryptophan synthase, in which Ser, Ala, Val or Gly was substituted for each of the three Cys residues, were analyzed using calorimetry. The mutants were less stable than the wild type, indicating that Cys residues contribute greatly to the stability of the alpha subunit. In most cases, a large decrease in denaturation enthalpy was observed, compensated for by the denaturation entropy to a major extent. The extent of changes in the denaturation Gibbs energy and denaturation enthalpy varied greatly depending on both substituting residues and positions. Of all the mutant proteins, the Cys154Ser mutant showed the greatest decrease in denaturation enthalpy; its denaturation enthalpy was half that of the wild type, and was considerably repaired by adding a ligand of the alpha subunit. Because the enthalpy of ligand binding to Cys154Ser in the native state did not change, it seems that the decrease in the denaturation enthalpy of Cys154Ser and its recovery by ligand binding are caused by conformational changes in the denatured state due to the mutation.
为了阐明埋藏在蛋白质内部的半胱氨酸残基在构象稳定性中的作用，利用量热法分析了大肠杆菌色氨酸合成酶突变体α亚基（其中三个半胱氨酸残基分别被丝氨酸、丙氨酸、缬氨酸或甘氨酸取代）的变性热力学性质。这些突变体比野生型更不稳定，表明半胱氨酸残基对α亚基的稳定性有很大贡献。在大多数情况下，观察到变性焓大幅下降，在很大程度上由变性熵补偿。变性吉布斯自由能和变性焓的变化程度因取代残基和位置的不同而有很大差异。在所有突变蛋白中，Cys154Ser突变体的变性焓下降最大；其变性焓是野生型的一半，通过添加α亚基的配体可得到显著修复。由于配体在天然状态下与Cys154Ser结合的焓没有变化，似乎Cys154Ser变性焓的下降及其通过配体结合的恢复是由突变导致的变性状态下的构象变化引起的。
