Multiscale study of the phenotypic consequences of protein folding intermediates in dihydrofolate reductase

二氢叶酸还原酶中蛋白质折叠中间体表型后果的多尺度研究

• 批准号: 9468581
• 负责人: Evgeny Serebryany
• 金额: $ 5.87万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9468581
• 关键词: Adopted; Affect; Amino Acid Sequence; Ankyrins; Antibiotic Resistance; Antibiotics; Bacterial Proteins; Base Sequence; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biophysics; Biotechnology; Case Study; Cataract; Cells; Chromatography; Column Chromatography; Complement; Computer Simulation; Cystic Fibrosis; Data; Defect; Deposition; Deuterium; Dihydrofolate Reductase; Disease; Enzymes; Escherichia coli; Fractionation; Genomics; Genotype; Goals; HIV; Hydrogen; Hydrophobic Interactions; Hydrophobicity; Improve Access; In Vitro; Intervention; Investigation; Kinetics; Lead; Libraries; Link; Literature; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Medical; Messenger RNA; Methods; Molecular Chaperones; Molecular Conformation; Monte Carlo Method; Mutation; Outcome; Paper; Parkinson Disease; Pathway interactions; Phage Display; Pharmaceutical Preparations; Phenotype; Population; Property; Proteins; Quality Control; RNA; RNA-Directed DNA Polymerase; Role; Serpins; Shapes; Site; Somatic Mutation; Structure; TP53 gene; Techniques; Temperature; Testing; Therapeutic Intervention; Therapeutic antibodies; Translating; Two-Hybrid System Techniques; Variant; Work; antimicrobial drug; bacterial fitness; base; cellular targeting; crosslink; design; experimental study; fitness; gain of function; high throughput screening; in vivo; inhibitor/antagonist; loss of function; mutant; mutation screening; nanobodies; polypeptide; protein folding; protein function; protein protein interaction; protein structure; resistance mutation; scaffold; screening; simulation

Native atomic structures of many proteins are now known, but they are often just the tip of the iceberg: there are intermediate states on the way to the native state and off-pathway kinetic traps and oligomers. Intermediates have important biological consequences. Many are aggregation-prone and associated with protein deposition diseases, from Parkinson's and ALS to serpin deficiency. Intermediates are targeted by the cellular protein quality control machinery – which can lead to disease, as in cystic fibrosis. Mutations linked to congenital diseases (from ALS to Creutzfeldt-Jakob to cataracts) or somatic mutations in sporadically arising diseases (p53- deficient cancers) often shift specific proteins toward intermediate conformations. Conversely, intermediates close to the native state are targets of remarkably successful drugs. Thus, nonnucleoside inhibitors of HIV reverse transcriptase bind to a pocket absent in that protein's native structure; the drug itself induces or selects this off-native conformation. Finally, intermediates have a critical impact on biotechnology – for example, for stability and storage of therapeutic antibodies. Despite its medical significance, the space of protein intermediates remains largely unexplored. Most have low stability and tend to oligomerize or aggregate, making structure determination very challenging. The most fruitful approach has been to find variants (mutants) that stabilize a given intermediate enough for structural investigation, but this requires good mutational screens. Screens in vitro or in silico can reveal intermediates, yet at low throughput; in vivo screens are high-throughput, yet they can detect intermediates only indirectly. However, in vitro studies of protein-protein interactions (PPI) have advanced greatly in recent years due to high- throughput screening techniques. This project will integrate rapid in silico unfolding simulations, in vitro screening methods adapted from the PPI field, and in vivo measurements of bacterial fitness to investigate intermediates of an essential bacterial enzyme, dihydrofolate reductase (DHFR), which is the target of many antibiotics and the locus of many antibiotic-resistance mutations. This project has three goals. First, to detect populated intermediates in a large library of DHFR variants, combining atomistic Monte Carlo simulations and hydrophobicity fractionation of the protein library in vitro. This requires adapting a “display” method from the PPI field: barcoding each protein molecule with RNA so as to identify the fractionated variants via barcode sequencing. Second, to distinguish and stabilize intermediates populated by mutants (and thus perhaps inducible in the wild-type protein as well) using a library of conformationally selective binding partners, such as nanobodies. Third, to evaluate the effect of distinct DHFR intermediates on the fitness of E. coli cells. Accomplishing these goals will map accessible DHFR intermediates that could be new antibiotic targets. It will also provide a much needed case study on the role of protein folding intermediates on all scales: from atomistic details to fitness effects in populations.

现在已知许多蛋白质的本地原子结构，但它们通常只是冰山一角：有 中间状态在前往本地状态和越野动力学陷阱和低聚物的途中。 具有重要的生物学后果。许多易于聚集，并且与蛋白质沉积有关 从帕金森氏症和ALS到Serpin缺乏症的疾病。中间体是由细胞蛋白靶向的 质量控制机制 - 可能导致疾病，如囊性纤维化。与先天性有关的突变 疾病（从ALS到Creutzfeldt-Jakob再到白内障）或偶发性疾病中的体细胞突变（P53-- 缺乏癌症）通常将特定的蛋白质转移到中间会议上。相反，中间体 接近本地状态的是非常成功的药物的靶标。那就是艾滋病毒的非核苷抑制剂 逆转录酶结合该蛋白质天然结构中不存在的口袋；药物本身诱导或选择 这个偏僻的构象。最后，中间体对生物技术有关键影响 - 例如 热抗体的稳定性和存储。 尽管具有医学意义，但蛋白质中间体的空间仍然在很大程度上出乎意料。大多数人的水平很低 稳定性并倾向于将寡聚或聚集起来，从而使结构确定非常挑战。最富有成果的 方法是找到稳定给定中间的变体（突变体），以进行结构 调查，但这需要良好的突变屏幕。体外或计算机中的屏幕可以揭示中间体，但 在低通量时；体内筛选是高通量的，但它们只能间接检测中间体。 然而，近年来，由于高 - 吞吐量筛查技术。该项目将迅速集成到硅的展开模拟中，体外筛选 从PPI场和细菌适应性的体内测量中改编的方法以研究中间体 基本细菌酶，二氢叶酸还原酶（DHFR），这是许多抗生素和 许多抗生素抗性突变的基因座。 该项目有三个目标。首先，要检测大型DHFR库中的填充中间体， 在体外结合了蒙特卡洛模拟和疏水性分馏。这 需要从PPI字段适应“显示”方法：用RNA将每个蛋白质分子进行条形码，以便 通过条形码测序识别分级变体。第二，以区分和稳定中间体 使用突变体（因此也可能在野生型蛋白中诱导）的填充 构象选择性结合伴侣，例如纳米构造。第三，评估不同DHFR的效果 大肠杆菌细胞的适应性中间。实现这些目标将绘制可访问的DHFR中间体 那可能是新的抗生素靶标。它还将提供急需的案例研究，以了解蛋白质折叠的作用 所有尺度上的中间体：从原子细节到人口的适应性效应。