Rodríguez Montserrat, Benito Antoni, Ribó Marc, Vilanova Maria
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Spain.
FEBS J. 2006 Mar;273(6):1166-76. doi: 10.1111/j.1742-4658.2006.05141.x.
It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. At 29 degrees C in a 20% (v/v) ethanol buffer, a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mm at 29 degrees C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease, a decrease in the temperature shifts the monomer-dimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model, an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.
先前已有报道称，一种人胰腺核糖核酸酶变体（即PM8）的结构通过N端结构域的交换形成二聚体，尽管在水溶液中它主要以单体形式存在。首先，我们研究了有利于该变体二聚化的溶液条件。在29℃的20%（v/v）乙醇缓冲液中，发现相当一部分蛋白质以二聚体形式存在，且未出现更高的寡聚体。通过尺寸排阻色谱法分离出这种二聚体，并对二聚化过程进行了研究。这种二聚体形式在29℃时的解离常数为5 mM。对二聚化过程对温度的依赖性分析表明，与牛胰腺核糖核酸酶不同，温度降低会使单体 - 二聚体平衡向二聚体形式移动。我们还表明，在二聚化过程之前，可交换结构域不会先从蛋白质主体上解离。我们的结果提出了一种与同源牛胰腺核糖核酸酶二聚化所假定的模型不同的PM8二聚化模型。在这个模型中，首先形成一个开放界面，然后亚基间相互作用将铰链环稳定在一种构象中，这种构象完全将未交换和交换二聚体之间的平衡转移到后者。
