Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases
Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases
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DOI：
10.1042/bj20030323
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发表时间：
2003-09-14
影响因子：
4.1
通讯作者：
LiCata, VJ
中科院分区：
文献类型：
--
作者：
Karantzeni, I;Ruiz, C;LiCata, VJ
用差示扫描量热法和圆二色谱法研究了水生栖热菌（Taq聚合酶）和大肠杆菌（Pol 1）的I型DNA聚合酶的热变性。全长蛋白质是包含聚合酶结构域、校正结构域（在Taq中无活性）和5'核酸酶结构域的单多肽链。去除5'核酸酶结构域产生Pol I和Taq的'大片段'结构域，分别称为Klenow和Klentaq。虽然Taq聚合酶的高温稳定性是众所周知的，但其热变性以前从未被直接检测过。这两种聚合酶的热变性是不可逆的，排除了严格的热力学分析。然而，聚合酶的比较熔融行为产生关于结构域结构、结构域相互作用以及两种聚合酶的稳定力的相似性和差异的信息。在差示扫描量热法中，Klenow和Klentaq变性为单峰，在pH 9.5时的熔融温度Tm分别为37和100 ℃。发现这两种全长聚合酶都由两个热力学解折叠结构域组成，每个结构域的5'核酸酶结构域分别解链。Taq的5'核酸酶结构域在Klentaq结构域之前10 ℃变性为单独的峰。Pol I的5'核酸酶结构域的解链与Klenow片段重叠。5'核酸酶结构域的存在使大片段在Pol 1中稳定，但使其在Taq中不稳定。Klentaq和Klenow变性对pH和甲醇的依赖性非常相似，表明疏水力和质子化作用稳定蛋白质的相似性。通过CD监测的熔融产生稍低的T-m值，但几乎相同的范特霍夫焓Δ H值，与两态展开一致，随后是不可逆的动力学步骤。与Arrhenius形式主义的变性扫描速率依赖性的分析估计Klentaq的不可逆变性的动力学障碍是显着高于Klenow。
Thermal denaturations of the type I DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol I and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T-m of 37 and 100 degreesC respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degreesC before the Klentaq domain . Melting of the 5' nuclease domain of Pol I overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T-m values, but almost identical van't Hoff enthalpy DeltaH values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.