Developing and Testing a Novel Geometric Model of Protein Adaptation

描述(申请人提供)：抗生素耐药性的进化通常由单一蛋白质中的错义突变介导(例如，抗叶酸耐药性突变经常发生在二氢叶酸还原酶中，喹啉耐药突变经常发生在DNA旋转酶中，利福霉素耐药突变经常发生在RNA聚合酶中)。这一事实引起了人们对蛋白质进化机制细节的关注：个体突变如何影响蛋白质的结构和功能，从而使病原微生物在药物存在的情况下存活？β-内酰胺类抗生素(如青霉素和头孢菌素类)占世界抗生素市场的65%[65]，而透射电子显微镜丝氨酸β-内酰胺酶是临床上质粒介导的β-内酰胺类抗生素耐药的主要来源[]。TEMb-内酰胺酶活性的生化和生物物理决定因素可通过实验获得[例如2，7]，这使其成为剖析蛋白质进化机制过程的理想模型系统。目前的提议是使用一种新的蛋白质进化几何模型来实现这一点。这项工作同时推进了进化遗传学领域，该领域现在正开始从自然选择证据的统计检验转向分子机制问题[4]。最近的工作强调了这样一个事实：蛋白质要想成功，就必须履行其功能(例如，酶必须催化它们的化学反应)，但同时，它在折叠稳定性、聚集和降解潜力等结构特征方面也处于稳定选择(即必须在狭窄的容差范围内)。此外，大多数错义突变表现出多效性，这意味着它们扰乱了不止一个这样的特征。这些生物物理和生化方面的考虑激发了蛋白质进化的图景，其中适应是一系列突变，每一种突变都具有净益处，通过大幅改善某些特征，同时适度降低其他特征[5]。该模型将蛋白质进化分解为其最近端的力学成分，形式上类似于RA Fisher首先提出的适应几何模型[6]。在目前的提议中，这一理论被扩展到允许根据它们对有机体适应性的影响来推断突变的作用机制。这一理论创新很重要，因为衡量突变对健康的影响往往比识别它们的生化和生物物理作用模式更实用。然后，这一理论将被应用于TEMb-内酰胺酶的一组新的突变，其对耐药性的影响将被表征为适应性的替代。最后，利用TEMb-内酰胺酶的实验可操作性，使用这一理论做出的关于这些b-内酰胺酶突变的预测将通过对这些相同突变机制的自下而上、生物化学和生物物理表征来验证。

DESCRIPTION (provided by applicant): Antibiotic resistance evolution is commonly mediated by missense mutations in a single protein (e.g. antifolate resistance mutations are often in dihydrofolate reductase, quinoline resistance mutations are often in DNA gyrase, rifamycin resistance mutations are often in RNA polymerase). This fact draws attention to the mechanistic details of protein evolution: how do individual mutations influence protein structure and function so as to allow pathogenic microbes to survive in the presence of the drug? b-lactam antibiotics (e.g. penicillin and the cephalosporins) represent 65% of the world antibiotic market [65], and the TEM serine b-lactamases are the chief source of clinical, plasmid mediated b-lactam resistance [64]. The biochemical and biophysical determinants of TEM b-lactamase activity are experimentally accessible [e.g. 2, 7], making this an ideal model system in which to dissect the mechanistic process of protein evolution. The present proposal is to use a novel, geometric model of protein evolution to do just this. This work simultaneously advances the field of evolutionary genetics, which is now beginning to move from statistical tests for evidence of natural selection to questions of molecular mechanism [4]. Recent work has highlighted the fact that to be successful a protein must perform its function (e.g. enzymes must catalyze their chemical reactions) but simultaneously it is under stabilizing selection (i.e. must fall within narrow tolerances) for structural traits such as folding stability, aggregation and degradation potentials and likely others. Moreover, most missense mutations exhibit pleiotropy, meaning that they perturb more than one such trait. These biophysical and biochemical considerations motivate a picture of protein evolution in which adaptation is a succession of mutations, each with net beneficial effect that act by substantially improving some traits while modestly degrading others [5]. This model decomposes protein evolution into its mechanistically most proximal components, and is formally analogous to a geometric model of adaptation first proposed by RA Fisher [6]. In the present proposal, this theory is extended to allow inference into mutational mechanisms of action based on their effect on organismal fitness. This theoretical innovation is important because measuring mutational effects on fitness is often far more practical than is the identification of their biochemical and biophysical mode of action. This theory will then be applied to a novel panel of mutations in TEM b-lactamase, whose effect on drug resistance will be characterized as a proxy for fitness. Finally, capitalizing on the experimental tractability of TEM b- lactamases, predictions made about these b-lactamase mutations using this theory will be validated via the bottom-up, biochemical and biophysical characterization of the mechanism of these same mutations.