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

项目摘要/摘要 同一蛋白质中突变位点之间的非相加相互作用可以决定选择性可及性 通过序列空间的可选突变路径，因此代表了潜在的 适应性蛋白质进化中的偶然性。如果突变的适合度效应依赖于基因 背景技术过去累积的替换历史可能会影响可允许的突变集合 因此，在未来，特别的适应结果可能取决于祖先的起点。为 因此，物种A可能比物种B(和物种B)更容易获得特定的适应性解决方案 反之亦然)仅仅是由于遗传背景的进化差异。这个项目的目标是在实验中 检测这种偶然性在心肌细胞跨膜Na+，K+-ATPase蛋白适应性进化中的作用 眼镜蛇。具体地说，我将结合定点突变实验和祖先蛋白质 复活研究Na~+，K~+-ATPase靶部位不敏感性的分子基础，蛋白质的变化 调节对心脏糖苷类药物的抵抗的功能。在几个不同的蛇家族中，对 在捕食有毒蟾蜍的物种中，心脏糖苷已经独立进化。在不同的物种之间 吃蟾蜍的蛇，以及进化出对心脏的抵抗力的许多其他动物类群 糖苷，靶部位不敏感的Na+，K+-ATPase归因于不同组合的分歧， 聚合，和平行的氨基酸替换，提供了丰富的比较数据。使用祖先 结合基于定点突变的蛋白质工程方法的蛋白质复活，即 将比较原因替换在特定于支系的祖先背景上的功能效果 更古老的祖先的背景，由所有的支系共享。该项目的具体目标是：(1) 确定导致蛇类对心脏糖苷类药物产生抵抗力的替代基因，以及(2) 确定这些致病替换的功能改变效应依赖于什么程度 它们发生的遗传背景。共同完成目标1和目标2将揭示分子基础 这是一项关键的生理创新，并将提供对这种创新的途径的一般见解 进化。这项拟议的研究涉及内布拉斯加州大学林肯分校(UNL)的Jay Storz博士的培训。 斯托兹博士是蛋白质进化领域的领军人物，以采取多学科方法获得 对进化机制有了更全面的理解。为此提出的培训计划 研究将为我的研究体系增加一个强大的进化生物学成分，结果是 显著增加我将能够解决的问题的广度和我将能够形成的协作 在今后的工作中。此外，UNL是博士后培训的理想环境，因为它提供了 为职业发展提供大量关键资源和高生产力和支持性的环境。

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.