A High-Resolution Luminescent Assay For Rapid And Continuous Monitoring Of Protein Translocation Across Biological Membranes

BioRxiv : the Preprint Server for Biology
Gonçalo C PereiraIan Collinson

Abstract

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises 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 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 rigour unattainable through classical methods.

Related Concepts

Luciferases
Inhibitors
Protein Translocation
Protein Transport
Pharmacologic Substance
Monitoring - Action
Developmental Arithmetic Disorder
Luminescent Measurements
In Vivo
Nuclear Translocation

Related Feeds

BioRxiv & MedRxiv Preprints

BioRxiv and MedRxiv are the preprint servers for biology and health sciences respectively, operated by Cold Spring Harbor Laboratory. Here are the latest preprint articles (which are not peer-reviewed) from BioRxiv and MedRxiv.

Related Papers

Annual Review of Biochemistry
N Pfanner, W Neupert
BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
U Wienhues, W Neupert
Biochimica Et Biophysica Acta
M Mori, K Terada
© 2021 Meta ULC. All rights reserved