A high-resolution luminescent assay for rapid and continuous monitoring of protein translocation across biological membranes

Goncalo De Castro Pereira, William Allen, Dan Watkins, Lisa Buddrus, Dylan Noone, Xia Liu, Andy Richardson, Ian Collinson*, Agnieszka Chacinska

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
208 Downloads (Pure)

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 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.
Original languageEnglish
Pages (from-to)1689-1699
Number of pages11
JournalJournal of Molecular Biology
Volume431
Issue number8
Early online date13 Mar 2019
DOIs
Publication statusPublished - 5 Apr 2019

Structured keywords

  • BrisSynBio
  • Bristol BioDesign Institute

Keywords

  • bacterial Sec system
  • live assay
  • mitochondrial protein import
  • NanoLuc
  • protein translocation
  • synthetic biology

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