- Main
Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins
Abstract
Monoubiquitination of proteins governs diverse physiological processes, and its dysregulation is implicated in multiple pathologies. The difficulty of preparing sufficient material often complicates the biophysical studies of monoubiquitinated recombinant proteins. Here we describe a robust avidity-based method that overcomes this problem. As a proof-of-concept, we produced milligram quantities of two monoubiquitinated targets, Parkinson's protein α-synuclein and ESCRT-protein ALIX, using NEDD4-family E3 ligases. Monoubiquitination hotspots were identified by quantitative chemical proteomics. Using FRAP and dye-binding assays, we uncovered strikingly opposite effects of monoubiquitination on the phase separation and fibrillization properties of these two amyloidogenic proteins, reflecting differences in their intermolecular interactions, thereby providing unique insights into the impact of monoubiquitination on protein aggregation.
Many UC-authored scholarly publications are freely available on this site because of the UC's open access policies. Let us know how this access is important for you.
Main Content
Enter the password to open this PDF file:
-
-
-
-
-
-
-
-
-
-
-
-
-
-