Properties of the Denatured States of Thermophilic versus Mesophilic Proteins

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的知识产权：该项目解决了为什么某些蛋白质在非常高的温度下保持稳定。 生物体中的蛋白质必须折叠成特定形状才能发挥作用。 正确折叠的状态被称为“天然”状态，而无序的未折叠状态被称为“变性”状态。 几条热力学证据导致了这样一种假设，即某些蛋白质的变性状态的性质是决定蛋白质在高温下稳定性的关键。 该项目通过检查同源蛋白质对的变性状态的特性，以几种不同的方式实验性地测试这一假设，其中蛋白质对的一个成员是高度温度稳定的（嗜热蛋白质），而另一个成员很容易随温度展开（嗜温蛋白质）。该项目的一个中心焦点是比较嗜热与嗜温DNA聚合酶的变性状态。 DNA聚合酶是复制DNA的蛋白质。 Klenow和Klentaq DNA聚合酶，来自E.大肠杆菌和水生栖热菌分别具有几乎相同的天然结构，但Klentaq聚合酶在比完全变性Klenow聚合酶高50摄氏度的温度下稳定。 热力学研究表明，Klentaq聚合酶的变性状态比Klenow的变性状态显着更紧凑和/或更少的动态，尽管它们的天然状态是高度同源的。 本计画将广泛地描述这两种不同聚合酶的整体结构（大小和形状）、折叠时的脱水和变性状态的动力学，然后将开始用其他类似的蛋白质对来检验这些结果的普遍性。这项工作的智力价值包括它将直接有助于解决“蛋白质折叠问题”。 了解蛋白质如何折叠到它们的天然状态，以及为什么有些蛋白质在能量上是稳定的，而另一些则不是，构成了生物学中最重要的问题之一，从基础生物化学到蛋白质工程都有影响。 理解蛋白质的变性状态是“问题的一半”，然而迄今为止，近90%的蛋白质折叠问题的工作只研究了蛋白质的天然状态。 该项目将是嗜热和嗜温蛋白质对的变性状态之间的第一个直接结构比较，它将直接，实验性地将这些变化与折叠的能量学和热力学联系起来。 该项目的初步数据已经表明，两种变性状态之间可能存在显着的大小差异，该项目将是第一个直接的结构观察和表征这种差异。 这种差异将改变目前广泛持有的观点，即变性状态的大小是序列无关的，仅仅是蛋白质链长度的函数。 因此，该项目旨在利用热力学和聚合物结构分析方法，为任何蛋白质系统的变性状态提供最完整的结构-热力学观点之一。 此外，设计一套易于应用的方法来快速确定其他嗜热-嗜温蛋白质对的变性状态的全局性质，以便其他研究人员能够更快地进行类似的比较，也将是这项工作的智力优势。更广泛的影响：这项工作的更广泛的影响和教育目标包括：1）在路易斯安那州立大学的普通生物化学和物理生物化学课程中引入蛋白质变性状态的最新观点，以帮助减少教科书信息与真实的研究之间的差距; 2）本科生研究人员的重要整合到项目中，3）为普通观众开发一个关于蛋白质折叠的戏剧，具有很强的科学内容，将与路易斯安那州立大学传播学系合作制作。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)Intellectual Merit: This project addresses why certain proteins remain stable at very high temperatures. Proteins in living organisms must fold into specific shapes in order to function. The properly folded state is called the "native" state, while the disorganized, unfolded state is called the "denatured" state. Several lines of thermodynamic evidence have led to the hypothesis that the properties of the denatured states of some proteins hold the key to how stable the proteins are to high temperature. This project experimentally tests this hypothesis in several different ways by examining the properties of the denatured states of pairs of homologous proteins, where one member of the protein pair is highly temperature stable (the thermophilic protein), while the other member of the pair is easily unfolded with temperature (the mesophilic protein). A central focus of the project is to compare the denatured states of a thermophilic versus a mesophilic DNA polymerase. DNA polymerases are proteins that replicate DNA. Klenow and Klentaq DNA polymerases, from E. coli and Thermus aquaticus, respectively, share an almost identical native structure, yet Klentaq polymerase is stable at temperatures 50 degrees Celcius higher than those that fully denature Klenow polymerase. Thermodynamic studies suggest that the denatured state of Klentaq polymerase is significantly more compact and/or less dynamic than the denatured state of Klenow, despite the fact that their native states are highly homologous. This project will extensively characterize the global structure (size and shape), dehydration upon folding, and dynamics of the denatured states of these two different polymerases, and then will begin to test the generality of these results with other similar pairs of proteins.The intellectual merit of this work includes the fact that it will directly contribute to helping solve the "protein folding problem". Understanding how proteins fold to their native states, and why some proteins are energetically stable while others are not, constitutes one of the most important questions in biology, with impact from fundamental biochemistry to protein-engineering. Understanding the denatured state of proteins is "half the problem", yet nearly 90% of all work on the protein folding problem has thus far examined only the native states of proteins. This project will be the first direct structural comparison between the denatured states of a thermophilic and mesophilic protein pair, and it will directly, experimentally link those changes to the energetics and thermodynamics of folding. The preliminary data for this project already indicate that there may be significant size differences between the two denatured states, and this project would be the first direct structural observation and characterization of such differences. Such differences would alter the current widely held view that denatured state size is sequence independent and solely a function of protein chain length. This project thus aims to utilize thermodynamics and methods of polymer structural analysis to provide one of the most integrated structural-thermodynamic views of the denatured state for any protein system. Further, the design of an easily applicable suite of methods for quick determinations of the global properties of the denatured states of other thermophilic-mesophilic protein pairs, so that other researchers will be able to more quickly perform similar comparisons, will also be an intellectual benefit of this work.Broader Impact: The broader impact and educational goals of this work include: 1) Introducing a more up-to-date view of the denatured state of proteins into the General Biochemistry and Physical Biochemistry courses at Louisiana State University to help decrease the gap between textbook information and real research; 2) Significant integration of undergraduate researchers into the project and 3) Development of a play for general audiences about protein folding, with strong scientific content, to be produced in collaboration with the Communication Studies Department at the Louisiana State University.