Epistasis as a source of contingency in the adaptive evolution of protein function

项目总结/摘要 同一蛋白质中突变位点之间的非加性相互作用可以决定蛋白质的选择性可及性。 通过序列空间的替代突变途径，因此代表了一个潜在的来源， 适应性蛋白质进化的偶然性。如果突变的适应性效应依赖于遗传 在背景技术中，过去置换的累积历史可能影响允许突变的集合 因此，特定的适应性结果可能取决于祖先的起点。为 因此，一个特定的自适应解决方案可能对物种A比对物种B更容易获得（反之亦然 相反，这仅仅是由于遗传背景的差异。这个项目的目标是实验性地 测试这种偶然性在跨膜Na+，K+-ATP酶蛋白的适应性进化中的作用， 游蛇具体地说，我将联合收割机定点突变实验与祖先蛋白结合起来， 复活以检查Na+，K+-ATP酶靶位点不敏感性的分子基础，即蛋白质的变化 介导对强心苷的抗性的功能。在几种不同的蛇类中， 强心苷在捕食有毒蟾蜍的物种中独立进化。在不同种类的 吃蟾蜍的蛇，以及许多其他动物类群，已经进化出对心脏病的抵抗力， 糖苷，Na+，K+-ATP酶的靶位点不敏感性可归因于不同的， 收敛的和平行的氨基酸取代，提供了丰富的比较数据。使用祖先 蛋白质复活与基于定点诱变的蛋白质工程方法相结合， 将比较致病替换对分支特定祖先背景和 一个更古老的祖先的背景，由所有分支共享。该项目的具体目标是（1） 鉴定在眼镜蛇中赋予对强心苷抗性的致病取代，以及（2） 确定这些致使性替代的功能改变效应依赖于 它们发生的遗传背景。共同实现目标1和2将揭示分子基础 一个关键的生理创新，并将提供一般的见解，通过这种创新的途径， 进化拟议的研究涉及与内布拉斯加大学林肯分校（UNL）的Jay Storz博士一起进行培训。 博士Storz是蛋白质进化领域的领导者，以采取多学科方法获得 更全面地理解进化的机制。为此提出的培训计划 研究将增加一个强大的进化生物学组成部分，我的研究剧目，因此， 大大增加我能够解决的问题的广度和我能够形成的合作 在今后的工作中。此外，UNL代表了博士后培训的理想环境，因为它提供了 职业发展的众多关键资源和高效和支持性的环境。

Project Summary/Abstract Nonadditive interactions between mutant sites in the same protein can dictate the selective accessibility of alternative mutational pathways through sequence space, and therefore represent a potential source of contingency in adaptive protein evolution. If the fitness effects of mutations are dependent on genetic background, the accumulated history of substitutions in the past may influence the set of allowable mutations in the future, and particular adaptive outcomes may therefore be contingent on ancestral starting points. For this reason, a particular adaptive solution may be more accessible to species A than to species B (and vice versa) simply due to evolved differences in genetic background. The goal of this project is to experimentally test the role of such contingency in the adaptive evolution of the transmembrane Na+,K+-ATPase protein in colubroid snakes. Specifically, I will combine site-directed mutagenesis experiments with ancestral protein resurrection to examine the molecular basis of target-site insensitivity of Na+,K+-ATPase, a change in protein function that mediates resistance to cardiac glycosides. In several different families of snakes, resistance to cardiac glycosides has evolved independently in species that prey on toxic toads. Among different species of toad-eating snakes, and among numerous other animal taxa that have evolved resistance to cardiac glycosides, target-site insensitivity of Na+,K+-ATPase is attributable to various combinations of divergent, convergent, and parallel amino acid substitutions, providing a rich body of comparative data. Using ancestral protein resurrection in conjunction with a protein engineering approach based on site-directed mutagenesis, I will compare the functional effects of causative substitutions on clade-specific ancestral backgrounds and on the background of a more ancient ancestor, shared by all clades. The specific aims of the project are to (1) identify the causative substitutions that confer resistance to cardiac glycosides in colubroid snakes and (2) to determine the extent to which the function-altering effects of these causative substitutions are dependent on the genetic context in which they occur. Together, accomplishing Aims 1 and 2 will reveal the molecular basis of a key physiological innovation and will provide general insights into the pathways by which such innovations evolve. The proposed research involves training with Dr. Jay Storz at University of Nebraska-Lincoln (UNL). Dr. Storz, a leader in the field of protein evolution, is well-known for taking a multi-disciplinary approach to gain a more complete understanding of the mechanisms governing evolution. The proposed training plan for this research would add a strong evolutionary biology component to my research repertoire, and as a result significantly increase the breadth of questions I will be able to address and collaborations I will be able to form in future work. Furthermore, UNL represents an ideal environment for postdoctoral training because it provides numerous key resources for career development and a highly productive and supportive environment.