A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes
Year: 2019
Type: article
Abstract: Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.
Source: Journal of Molecular Biology
Institutions: University of Bristol, BrisSynBio, University of Warsaw
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $2,576
Awards: G1015763213, G1280652415, Understanding mitochondrial protein import and membrane insertion., BrisSynBio: Bristol Centre for Synthetic Biology, G3810972741, G4494338184, G621933505, G7045001909, Deciphering the allosteric mechanism of protein translocation through membranes, Understanding mitochondrial protein import and membrane insertion.
OpenAlex ID: W2950266092