相关实验视频

Updated: Jun 8, 2025

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Published on: December 17, 2013

通过使用技术上有效的BPA光交联方法,分析线粒体转位酶在工作中的蛋白质-蛋白质相互作用

Edward Germany¹, Takuya Shiota¹

¹Frontier Science Research Center, University of Miyazaki, Kiyotake, Miyazaki, Japan.

Methods in enzymology

|November 2, 2024

概括

这项研究展示了使用p-benzoyl-L-phenylalanine (pBpa) 来绘制酵母体中线粒体转位酶复合体内的蛋白质相互作用的照片交叉链接. 这种方法在接近生理条件下的高空间分辨率捕捉蛋白质的近距离.

科学领域:

生物化学生物化学
分子生物学分子生物学
细胞生物学细胞生物学

背景情况:

蛋白质与蛋白质之间的相互作用对于细胞功能至关重要.
了解蛋白质复合体的空间组织对于破译它们的机制至关重要.
线粒体蛋白转位在蛋白质进口和细胞平衡中起着至关重要的作用.

研究的目的:

介绍和示范p-基-L-氨 (pBpa) 光交联方法用于分析蛋白质相互作用.
为了研究线粒体转位酶复合体内的复杂内亚单元-亚单元相互作用.
在近乎生理学的背景下检查转位酶通道-基质相互作用.

主要方法:

利用pBpa,一种具有光反应侧链的氨基酸,用于UV诱导的交联.
采用体内抑制器tRNA系统,将pBpa引入酵母蛋白.
在体内和器官内进行交叉连接实验,以分析Saccharomyces cerevisiae线粒体转位酶复合体内的蛋白质相互作用.

主要成果:

成功地将pBpa引入活酵母细胞中的标蛋白.
捕获并分析了线粒体转位酶复合体内的体内亚单元-亚单元相互作用.
在有机细胞环境中研究了转位酶通道-基质相互作用,提供了高空间分辨率.

结论:

pBpa光交联方法为绘制蛋白质接近度与氨基酸残留分辨率提供了强大的工具.
在体内和器官内交叉连接方法使得在近乎生理条件下的转位酶功能的强有力的分析.
这种方法有助于更深入地了解 mitochondrial translocases 等大型蛋白质复合物的结构和功能动态.

关键词:

线粒体蛋白质的进口照片交叉链接照片交叉链接蛋白质与蛋白质的相互作用姆 (TOM) 复合体是一个复合体.姆4040的时间.不自然的氨基酸是一种非自然的氨基酸.

更多相关视频

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Published on: July 19, 2021

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Published on: May 5, 2017

相关概念视频

Translocation of Proteins into the Mitochondria

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...

Energy to Drive Translocation

Energy to Drive Translocation

Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct...

Mitochondrial Precursor Proteins

Mitochondrial Precursor Proteins

Mitochondrial precursors are partially unfolded or loosely folded polypeptide chains. Newly synthesized precursors are inhibited from spontaneously folding into their native conformation by the cytosolic chaperones, heat shock proteins 70 (Hsp70), and mitochondrial import stimulation factors (MSFs). Precursors bound to MSFs are guided to the TOM70-TOM37 receptors, while precursors bound to Hsp70 chaperones are targetted to TOM20-TOM22 receptor complexes.
Most of the mitochondrial...

Mitochondrial Protein Sorting

Mitochondrial Protein Sorting

Mitochondria are double-membrane organelles of the eukaryotes involved in cellular metabolism, signaling, ATP synthesis, and programmed cell death. Each of these processes requires specific proteins and enzymes that must be correctly sorted to the right mitochondrial subcompartment for the proper functioning of the organelle.
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...

Protein Transport into the Inner Mitochondrial Membrane

Protein Transport into the Inner Mitochondrial Membrane

Nuclear encoded mitochondrial precursors are imported to the inner membrane in a multistep process involving two separate translocons, TIM22 and TIM23. TIM23 is a cation-selective pore that remains closed by the N terminal segment of the protein. Negative charges on the TIM23 act as a receptor for the incoming precursor, pulling the positively charged matrix-targeting sequence for peptide insertion and translocation.
Transport of mitochondrial precursors across the TIM23 channel is driven by...

Structure of Porins

Structure of Porins

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and metabolites across the membrane. Mitochondrial porin precursors contain conserved amino acid sequences called beta signals at their C-terminal. Beta signals have a motif of PoXGXXHyXHy (Po-Polar, X-Any amino acid, G-Glycine, Hy-LargeHydrophobic), which are crucial for precursor recognition to initiate precursor assembly. Beta-barrel...