Nov 1, 2018

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.

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Mentioned in this Paper

Biological Membrane
In Vivo
Protein Targeting to Mitochondrion
Inhibitors
Pharmacologic Substance
Nuclear Translocation
Monitoring - Action
Protein Transport
Luminescent Measurements
Screening Generic

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