Single-molecule studies of Sec-dependent protein translocation

Sec 依赖性蛋白质易位的单分子研究

• 批准号: 9374906
• 负责人: Christian Kaiser
• 金额: $ 19.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374906
• 关键词: ATP Hydrolysis; Amino Acid Sequence; Animals; Antibiotics; Antibodies; Automobile Driving; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Carrier Proteins; Cell membrane; Cell physiology; Cellular Membrane; Chemicals; Conflict (Psychology); Cytosol; Data; Defect; Dependence; Destinations; Development; Diabetes Mellitus; Disease; Drug Targeting; Endoplasmic Reticulum; Enzymes; Eukaryota; Foundations; Individual; Insulin; Ions; Lead; Light; Lipid Bilayers; Literature; Malignant Neoplasms; Measurement; Measures; Mechanics; Membrane; Molecular; Molecular Conformation; Molecular Machines; Motor; Movement; Nature; Operating System; Organism; Pathway interactions; Permeability; Power stroke; Process; Protein Export Pathway; Protein Secretion; Protein translocation; Proteins; Reporting; Resistance; Resolution; Role; Spectrum Analysis; Structure; Surface; System; Testing; Time; Variant; Virulence Factors; Work; drug development; experimental study; fighting; insight; laser tweezer; macromolecule; mechanical force; mechanical load; millisecond; molecular dynamics; mutant; nanometer; novel; pathogenic bacteria; polypeptide; protein transport; public health relevance; single molecule; solute; temporal measurement; tool

Project Summary Many essential proteins are inserted into membranes or secreted. Because they are synthesized in the cytosol, these proteins must cross a lipid bilayer to reach their destination and become functional. The majority of proteins bound for secretion or membrane insertion transits through the universally conserved Sec translocon. The Sec pathway allows proteins destined for export from the cytosol to cross the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in bacteria. The essential role of Sec-dependent translocation in many cellular pathways makes elucidation of the underlying molecular mechanisms an important task. Substrates of the Sec system include virulence factors and antibiotic-inactivating enzymes in bacteria, and range from insulin to antibodies in animals. Perturbations in the Sec-pathway can lead to numerous diseases, including cancer and diabetes. A mechanistic understanding of the translocation process can fuel the development of drugs that target this central cellular pathway. Sec-dependent protein translocation has been studied extensively with biochemical and structural approaches, characterizing many of its components. However, the dynamics of the process are not well understood. During translocation, the translocon channel must allow the polypeptide to move while maintaining a permeability barrier for ions and other solutes. How the channel interacts with polypeptide substrates to achieve these seemingly conflicting requirements is not known. Another key question that has remained unanswered is how the molecular machines that associate with the translocon convert chemical energy into the mechanical work that powers translocation. Many of the important outstanding questions concerning the Sec translocation system could be answered if it were possible to follow the passage of a protein through the channel in real-time with high spatial and temporal resolution, but this capability is not presently available. Single-molecule approaches, enabling observation and manipulation of individual macromolecules, have provided unprecedented insights into biological mechanisms. I propose to investigate the mechanisms of Sec- dependent protein translocation with optical tweezers. This approach enables us to unravel the mechanisms underlying Sec-dependent protein translocation. Our studies will yield new and exciting insights into the mechanisms employed by the translocation machinery to transport proteins out of the cytosol.

项目摘要 许多基本蛋白质被插入膜或分泌。因为它们是在细胞质中合成的，所以 这些蛋白质必须越过脂质双层才能到达目的地并起作用。大多数 与分泌或膜插入结合的蛋白质通过普遍保守的SEC转运。 SEC途径允许注定要从细胞质出口的蛋白质穿越内质网 真核生物中的膜和细菌中的质膜。依赖SEC的基本作用 许多细胞途径中的易位使基本分子机制阐明 重要任务。 SEC系统的底物包括毒力因子和抗生素灭活酶 细菌，从胰岛素到动物的抗体。 SEC-Pathway中的扰动可能会导致 许多疾病，包括癌症和糖尿病。对易位过程的机械理解 可以为针对该中央细胞途径的药物的发展加油。 已通过生化和结构方法对SEC依赖性蛋白易位进行了广泛的研究， 表征其许多组件。但是，该过程的动态尚不清楚。期间 易位，转运通道必须允许多肽在保持渗透性的同时移动 离子和其他溶质的障碍。通道如何与多肽底物相互作用以实现这些 看似相互矛盾的要求尚不清楚。尚未解决的另一个关键问题是如何 与转运量相关的分子机器将化学能转换为机械工作 这能力易位。有关SEC易位的许多重要问题 如果可以实时遵循蛋白质通过通道，可以回答系统 具有高空间和时间分辨率，但目前尚不可用。 单分子方法，使人对单个大分子的观察和操纵 提供了对生物学机制的前所未有的见解。我建议研究sec-的机制 依赖性蛋白质易位与光学镊子。这种方法使我们能够揭示机制 潜在的依赖SEC的蛋白质易位。我们的研究将产生新的令人兴奋的见解 易位机械采用的机制将蛋白质从细胞质中输送出来。