为 FRET 研究 Cdc48 底物加工制备定点荧光标记多泛素链。

Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing.

机构信息

Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA.

California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720.

出版信息

STAR Protoc. 2023 Dec 15;4(4):102659. doi: 10.1016/j.xpro.2023.102659. Epub 2023 Oct 26.

DOI:10.1016/j.xpro.2023.102659

PMID:37889757

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10630674/

Abstract

A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023)..

摘要

将多泛素化蛋白质从大分子复合物和膜中去除以进行随后的蛋白酶体降解的关键步骤是通过辅助因子 Ufd1/Npl4（UN）展开泛素部分，并将其插入 Cdc48 ATPase 中进行机械易位。在这里，我们提供了一个逐步协议，用于组装和纯化 Lys48 连接的泛素链，这些泛素链在特定的泛素部分被荧光标记，并允许在基于 FRET 的测定中监测 Cdc48-UN 复合物与多泛素的结合。有关此协议的使用和执行的完整详细信息，请参阅 Williams 等人。（2023 年）。