Permyakov Sergei E, Khokhlova Tatyana I, Nazipova Aliya A, Zhadan Andrey P, Morozova-Roche Ludmila A, Permyakov Eugene A
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia.
Proteins. 2006 Dec 1;65(4):984-98. doi: 10.1002/prot.21159.
The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a "phase diagram" in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA.
研究单一位点金属结合蛋白金属结合特性最普遍的方法，即在游离金属离子浓度-温度坐标中构建“相图”，已应用于马溶菌酶（EQL）。EQL有一个相对较强的钙结合位点，并表现出两个热转变，但其中只有一个是Ca(2+)依赖性的。已发现，基于蛋白质中金属和温度诱导的结构变化的最简单四态方案，可以充分描述EQL低温热转变（I）的Ca(2+)依赖性行为。通过实验确定了该方案的所有热力学参数，并用于构建pCa-温度空间中的EQL相图。将该相图与溶菌酶的紧密同源物α-乳白蛋白（α-LA）的相图进行比较，可以直观地看到这两种蛋白质热力学行为的差异。脱辅基EQL（转变I）的热稳定性与脱辅基α-LA（中温25℃）的热稳定性非常相似，而它们的Ca(2+)结合形式的热稳定性几乎无法区分。与α-LA相比，EQL的天然状态对Ca(2+)的亲和力低三个数量级，而其热解折叠状态（在I转变后）对钙的亲和力低约一个数量级（K = 15M(-1)）。第一次热转变后脱辅基溶菌酶状态的圆二色性研究表明，它与α-LA的熔融球状状态具有共同特征。
