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.

现在许多蛋白质的天然原子结构已为人所知，但它们通常只是冰山一角： 通往天然状态的中间状态以及非路径动力学陷阱和低聚物。中间体 具有重要的生物学后果。许多易于聚集并与蛋白质沉积相关 疾病，从帕金森病、ALS 到丝氨酸蛋白酶抑制剂缺乏症。中间体是细胞蛋白质的目标 质量控制机制——可能导致疾病，如囊性纤维化。与先天性相关的突变 疾病（从 ALS 到克雅氏病再到白内障）或偶发性疾病中的体细胞突变 (p53- 缺陷型癌症）通常会将特定蛋白质转变为中间构象。反之，中间体 接近天然状态的区域是非常成功的药物的目标。因此，HIV非核苷抑制剂 逆转录酶与该蛋白质天然结构中不存在的口袋结合；药物本身诱导或选择 这种非天然构象。最后，中间体对生物技术具有至关重要的影响——例如， 治疗抗体的稳定性和储存。 尽管具有医学意义，但蛋白质中间体的空间在很大程度上仍未被探索。大多数都低 稳定性并且倾向于低聚或聚集，使得结构确定非常具有挑战性。最有成果的 方法是找到足够稳定给定中间体的变体（突变体），用于结构 调查，但这需要良好的突变筛选。体外或计算机屏幕可以揭示中间体，但 低吞吐量时；体内筛选是高通量的，但它们只能间接检测中间体。 然而，由于高蛋白相互作用（PPI）的体外研究近年来取得了很大进展。 通量筛选技术。该项目将整合快速计算机展开模拟、体外筛选 采用 PPI 领域的方法以及细菌适应性的体内测量来研究中间体 一种重要的细菌酶二氢叶酸还原酶 (DHFR)，它是许多抗生素和药物的作用靶点 许多抗生素耐药性突变的位点。 该项目有三个目标。首先，为了检测大型 DHFR 变体库中的填充中间体， 结合原子蒙特卡罗模拟和体外蛋白质库的疏水性分级分离。这 需要采用 PPI 领域的“展示”方法：用 RNA 对每个蛋白质分子进行条形码编码，以便 通过条形码测序识别分级变体。二、区分和稳定中间体 使用突变体库填充突变体（因此也可能在野生型蛋白质中诱导） 构象选择性结合伴侣，例如纳米抗体。三、评估不同DHFR的效果 大肠杆菌细胞适应性的中间体。实现这些目标将绘制可利用的 DHFR 中间体 这可能是新的抗生素靶点。它还将为蛋白质折叠的作用提供急需的案例研究 所有尺度的中间体：从原子细节到群体的适应性效应。