Engineered disulfide crosslinking to measure conformational changes in the 26S proteasome

• Published in: Methods in enzymology Vol. 619; p. 145
• Main Authors: Reed, Randi G, Tomko, Jr, Robert J
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; Cross-Linking Reagents - chemistry; Disulfides - chemistry; Models, Molecular; Proteasome Endopeptidase Complex - chemistry; Proteasome Inhibitors - chemistry; Proteasome Inhibitors - pharmacology; Protein Conformation - drug effects; Protein Subunits - chemistry; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae Proteins - antagonists & inhibitors; Saccharomyces cerevisiae Proteins - chemistry; Ubiquitin; Disulfide crosslinking; 26S proteasome; Multisubunit complex; Conformational dynamics; Proteasome inhibitors; Conformation
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2018.11.006

The 26S proteasome is a multisubunit ATP-dependent peptidase complex mediating most regulated protein degradation in eukaryotes. The proteasome undergoes several coordinated conformational changes during catalysis that activate it for substrate processing and functionally couple distinct enzymatic activities during substrate degradation. Understanding the impact of substrate interactions and individual ATP binding events on these conformational changes is currently a major bottleneck in the study of proteasome function. Here, we describe a simple biochemical reporter based on engineered disulfide crosslinking for measuring the conformational distribution of the Saccharomyces cerevisiae 26S proteasome. We demonstrate its use to investigate the impact of ATP analogs and proteasome inhibitors on proteasome conformational equilibria. This reporter allows simultaneous and rapid comparison of multiple treatments or conditions on the steady-state conformational distribution of the proteasome and can be readily extended to the study of other multisubunit complexes for which multiple conformational states are known at near-atomic resolution.