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 许多必需的蛋白质被插入细胞膜或被分泌。因为它们是在细胞质中合成的， 这些蛋白质必须穿过脂质双层才能到达它们的目的地并发挥功能。大多数 结合用于分泌或膜插入的蛋白质通过普遍保守的Sec易位子转运。 Sec途径允许蛋白质从胞质溶胶中输出穿过内质网 真核生物的质膜和细菌的质膜。Sec-dependent的重要作用 在许多细胞途径中的易位使得阐明了潜在的分子机制， 重要的任务。Sec系统的底物包括毒力因子和抗生素失活酶 从胰岛素到动物体内的抗体。SEC途径的扰动可导致 许多疾病，包括癌症和糖尿病。对易位过程的机械理解 可以促进针对这一中心细胞通路的药物的开发。 SEC依赖的蛋白质易位已经用生物化学和结构方法进行了广泛的研究， 它的许多组成部分。然而，人们对这一进程的动态并不十分了解。期间 在易位中，易位子通道必须允许多肽移动，同时保持渗透性。 离子和其他溶质的屏障。通道如何与多肽底物相互作用以实现这些 似乎相互矛盾的要求是未知的。另一个尚未回答的关键问题是， 与易位子相关的分子机器将化学能转化为机械功 为易位提供动力关于Sec易位的许多重要的悬而未决的问题 如果能够实时跟踪蛋白质通过通道的情况， 具有高的空间和时间分辨率，但这种能力目前还不可用。 单分子方法能够观察和操纵单个大分子， 为生物学机制提供了前所未有的见解。我建议调查的机制，秒- 依赖蛋白质移位的光镊技术。这种方法使我们能够解开 潜在的Sec依赖性蛋白质易位。我们的研究将产生新的和令人兴奋的见解， 易位机制将蛋白质转运出胞质溶胶。