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.

许多蛋白质的天然原子结构现在已为人所知，但它们往往只是冰山一角：有 在通往自然状态的过程中的中间态，以及偏离路径的动力学陷阱和低聚物。中间体 具有重要的生物学后果。许多是易于聚集的，与蛋白质沉积有关 疾病，从帕金森氏症和肌萎缩侧索硬化症到丝氨酸缺乏症。中间体是细胞蛋白质的靶标。 质量控制机制--这可能导致疾病，如囊性纤维化。与先天性相关的突变 疾病(从ALS到CreutzFeldt-Jakob再到白内障)或零星出现的疾病的体细胞突变(P53- 缺陷癌症)通常使特定的蛋白质向中间构象转移。相反，中间体 靠近这个州的是非常成功的药物的靶标。因此，HIV的非核苷抑制剂 逆转录酶结合到蛋白质天然结构中缺失口袋；药物本身诱导或选择 这种非原生的构象。最后，中间体对生物技术有至关重要的影响--例如 治疗性抗体的稳定性和储存。 尽管蛋白质中间体具有重要的医学意义，但它在很大程度上仍未被探索。大多数人都有低血压 稳定性和倾向于齐聚或聚集，使得结构确定非常具有挑战性。最有成效的 方法一直是寻找变种(突变体)，以稳定给定的中间体足够的结构 调查，但这需要良好的突变筛查。体外或硅胶中的筛选可以显示中间体，但 在低吞吐量的情况下，体内筛选是高通量的，但它们只能间接检测中间体。 然而，蛋白质-蛋白质相互作用(PPI)的体外研究近年来取得了很大进展，这是因为蛋白质相互作用的高密度 吞吐量筛选技术。该项目将集成快速在电子计算机展开模拟，在体外筛选 从PPI领域改编的方法，以及体内细菌适合性的测量，以调查中间体 一种重要的细菌酶，二氢叶酸还原酶(DHFR)，它是许多抗生素和 许多抗生素耐药突变的基因座。 这个项目有三个目标。首先，为了检测DHFR变异体的大型文库中填充的中间体， 结合原子蒙特卡罗模拟和疏水性分级的蛋白质文库在体外。这 需要从PPI领域采用一种“显示”方法：用RNA对每个蛋白质分子进行条形化，以便 通过条形码测序鉴定分离的变异。第二，区分和稳定中间体 由突变体(因此也可能在野生型蛋白中诱导)使用 构象选择性结合伙伴，如纳米体。第三，评估不同DHFR的效果 中间体对大肠杆菌细胞的适合性。实现这些目标将绘制可访问的DHFR中间体 这可能是新的抗生素目标。它还将为蛋白质折叠的作用提供一个亟需的案例研究 所有尺度上的中间体：从原子化的细节到种群中的适应性效应。