EAGER: Identification of molecular parameters defining the fine line between thermostability and thermophilic properties

来自嗜热生物的酶通常更适合生物技术应用，因为它们具有热稳定性并且在高温下具有最佳活性。 该项目将使用新颖的实验和计算方法来定义这些非常有用的特性的分子基础。 最终，这些研究将为选择在高温下稳定且具有最佳活性的酶以及设计此类酶在生物技术中的应用提供重要信息。 该项目将有助于本科生和研究生的培训，并将纳入对公众的宣传工作。阐明使蛋白质在高温等极端条件下更加稳定的分子原理一直是大量研究的主题。 早期的研究表明，酶的嗜热变体具有更多的极性相互作用或更多的氢键或增加的疏水性相互作用、更好的疏水核心堆积或它们的某种组合。 其他研究表明，其中一些特性不适用于许多其他嗜温与嗜热的比较，并且蛋白质的更微妙和分布的效应和动态特性可能在获得嗜热性方面发挥重要作用。 基于序列的比较也未能解释嗜热性的基础，迄今为止还没有出现明确且统一的解释。 该项目旨在通过剖析蛋白质动力学和溶剂重组对酶配体相互作用的影响，确定将嗜热酶与其嗜温对应物分开的动态和热力学特性。 为此，将使用同一蛋白质（氨基糖苷-N3-核苷酸转移酶）的嗜热和嗜温变体；这些变体在一级序列上的差异很小。

Enzymes from thermophilic organisms are often more suitable for applications in biotechnology because they are thermostable and have optimal activities at high temperatures. This project will use novel experimental and computational approaches to define the molecular basis for these highly useful properties. Ultimately, these studies will provide important information for the selection of enzymes that are stable and optimally active at high temperatures and for the engineering of such enzymes for applications in biotechnology. This project will contribute to the training of undergraduate and graduate students and will be incorporated into outreach efforts to the public. The elucidation of molecular principles that render proteins more stable under extreme conditions such as high temperatures has been subject of a large body of research. Earlier work suggested that thermophilic variants of enzymes have more polar interactions or more H-bonds or increased hydrophobic interactions, better packing of hydrophobic core or some combination of them. Other studies showed that some of these properties were not applicable to many other mesophilic to thermophilic comparisons and more subtle and distributed effects and dynamic properties of proteins may play a significant role in attaining thermophilicity. Sequence-based comparisons also failed to explain the basis of thermophilicity and so far no clear and unifying explanation has emerged. This project aims to determine dynamic and thermodynamic properties that separate thermophilic enzymes from their mesophilic counterparts by dissecting the effects of protein dynamics and solvent reorganization on the enzyme ligand interactions. To do this, thermophilic and mesophilic variants of the same protein (aminoglycoside-N3-nucleotidyltransferase) will be used; these variants have minimal differences in the primary sequence.