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A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes
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 p...
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| Published in: | Journal of molecular biology 2019-04, Vol.431 (8), p.1689-1699 |
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| Main Authors: | , , , , , , , , |
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| cites | cdi_FETCH-LOGICAL-c517t-8d0b8b4816d34bd39aa89adece49475f6b8e9e93d4d297437eec82e7321e286e3 |
| container_end_page | 1699 |
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| container_title | Journal of molecular biology |
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| creator | Pereira, Gonçalo C. Allen, William J. Watkins, Daniel W. Buddrus, Lisa Noone, Dylan Liu, Xia Richardson, Andrew P. Chacinska, Agnieszka Collinson, Ian |
| description | 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.
[Display omitted]
•Conventional methods for monitoring protein translocation are laborious and discontinuous and lack kinetic detail.•A split NanoLuc system was adapted for real-time monitoring of protein translocation through the bacterial Sec system and the mitochondrial Tim23 complex.•The new assay reduces the timescale of data acquisition from days to minutes.•It produces high-quality results suitable for kinetic fitting and model derivation. |
| doi_str_mv | 10.1016/j.jmb.2019.03.007 |
| format | article |
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[Display omitted]
•Conventional methods for monitoring protein translocation are laborious and discontinuous and lack kinetic detail.•A split NanoLuc system was adapted for real-time monitoring of protein translocation through the bacterial Sec system and the mitochondrial Tim23 complex.•The new assay reduces the timescale of data acquisition from days to minutes.•It produces high-quality results suitable for kinetic fitting and model derivation.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2019.03.007</identifier><identifier>PMID: 30878481</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>bacterial Sec system ; Escherichia coli - metabolism ; Escherichia coli Infections - microbiology ; Escherichia coli Proteins - metabolism ; Humans ; live assay ; Luciferases - metabolism ; Luminescent Agents - metabolism ; Luminescent Measurements - methods ; Mitochondrial Membranes - metabolism ; mitochondrial protein import ; NanoLuc ; protein translocation ; Protein Transport ; Saccharomyces cerevisiae - metabolism ; Saccharomyces cerevisiae Proteins - metabolism ; SEC Translocation Channels - metabolism</subject><ispartof>Journal of molecular biology, 2019-04, Vol.431 (8), p.1689-1699</ispartof><rights>2019 The Authors</rights><rights>Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.</rights><rights>2019 The Authors 2019</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c517t-8d0b8b4816d34bd39aa89adece49475f6b8e9e93d4d297437eec82e7321e286e3</citedby><cites>FETCH-LOGICAL-c517t-8d0b8b4816d34bd39aa89adece49475f6b8e9e93d4d297437eec82e7321e286e3</cites><orcidid>0000-0001-9638-0615 ; 0000-0003-0524-7021 ; 0000-0002-2832-2568 ; 0000-0003-3825-5036 ; 0000-0002-3931-0503 ; 0000-0002-9513-4786</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,315,786,790,891,27957,27958</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30878481$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pereira, Gonçalo C.</creatorcontrib><creatorcontrib>Allen, William J.</creatorcontrib><creatorcontrib>Watkins, Daniel W.</creatorcontrib><creatorcontrib>Buddrus, Lisa</creatorcontrib><creatorcontrib>Noone, Dylan</creatorcontrib><creatorcontrib>Liu, Xia</creatorcontrib><creatorcontrib>Richardson, Andrew P.</creatorcontrib><creatorcontrib>Chacinska, Agnieszka</creatorcontrib><creatorcontrib>Collinson, Ian</creatorcontrib><title>A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>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.
[Display omitted]
•Conventional methods for monitoring protein translocation are laborious and discontinuous and lack kinetic detail.•A split NanoLuc system was adapted for real-time monitoring of protein translocation through the bacterial Sec system and the mitochondrial Tim23 complex.•The new assay reduces the timescale of data acquisition from days to minutes.•It produces high-quality results suitable for kinetic fitting and model derivation.</description><subject>bacterial Sec system</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Infections - microbiology</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Humans</subject><subject>live assay</subject><subject>Luciferases - metabolism</subject><subject>Luminescent Agents - metabolism</subject><subject>Luminescent Measurements - methods</subject><subject>Mitochondrial Membranes - metabolism</subject><subject>mitochondrial protein import</subject><subject>NanoLuc</subject><subject>protein translocation</subject><subject>Protein Transport</subject><subject>Saccharomyces cerevisiae - metabolism</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>SEC Translocation Channels - metabolism</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><recordid>eNp9kV9rFDEUxYModq1-AF8kj77MmH87kyAI61KtsKVS6nPIJHe2WWaSNZkpFPzwzXZr0Zc-5eGee3LP-SH0npKaEtp82tW7sasZoaomvCakfYEWlEhVyYbLl2hBCGMVk7w5QW9y3hFCllzI1-iEE9lKIekC_Vnhc7-9qa4gx2GefAx4M48-QLYQJrzK2dzhPiZ8ZfbeYRMcXscw-TDHOeOLGPwUkw9bHHv8M8UJfMDXyYQ8RGse7IxNMWf81cchbr01A76AsSsSyG_Rq94MGd49vqfo17ez6_V5tbn8_mO92lR2Sdupko50sivnNo6LznFljFTGgQWhRLvsm06CAsWdcEy1grcAVjJoOaPAZAP8FH05-u7nbgR3SJbMoPfJjybd6Wi8_n8S_I3exlvdiIZSJYvBx0eDFH_PkCc9-lLQMJQUpQfNqOINWQrOipQepQ-xE_RP31CiD9T0Thdq-kBNE64LtbLz4d_7njb-YiqCz0cBlJZuPSSdrYdgwfkEdtIu-mfs7wHzkKuo</recordid><startdate>20190405</startdate><enddate>20190405</enddate><creator>Pereira, Gonçalo C.</creator><creator>Allen, William J.</creator><creator>Watkins, Daniel W.</creator><creator>Buddrus, Lisa</creator><creator>Noone, Dylan</creator><creator>Liu, Xia</creator><creator>Richardson, Andrew P.</creator><creator>Chacinska, Agnieszka</creator><creator>Collinson, Ian</creator><general>Elsevier Ltd</general><general>Elsevier</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-9638-0615</orcidid><orcidid>https://orcid.org/0000-0003-0524-7021</orcidid><orcidid>https://orcid.org/0000-0002-2832-2568</orcidid><orcidid>https://orcid.org/0000-0003-3825-5036</orcidid><orcidid>https://orcid.org/0000-0002-3931-0503</orcidid><orcidid>https://orcid.org/0000-0002-9513-4786</orcidid></search><sort><creationdate>20190405</creationdate><title>A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes</title><author>Pereira, Gonçalo C. ; Allen, William J. ; Watkins, Daniel W. ; Buddrus, Lisa ; Noone, Dylan ; Liu, Xia ; Richardson, Andrew P. ; Chacinska, Agnieszka ; Collinson, Ian</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c517t-8d0b8b4816d34bd39aa89adece49475f6b8e9e93d4d297437eec82e7321e286e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>bacterial Sec system</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Infections - microbiology</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Humans</topic><topic>live assay</topic><topic>Luciferases - metabolism</topic><topic>Luminescent Agents - metabolism</topic><topic>Luminescent Measurements - methods</topic><topic>Mitochondrial Membranes - metabolism</topic><topic>mitochondrial protein import</topic><topic>NanoLuc</topic><topic>protein translocation</topic><topic>Protein Transport</topic><topic>Saccharomyces cerevisiae - metabolism</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>SEC Translocation Channels - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pereira, Gonçalo C.</creatorcontrib><creatorcontrib>Allen, William J.</creatorcontrib><creatorcontrib>Watkins, Daniel W.</creatorcontrib><creatorcontrib>Buddrus, Lisa</creatorcontrib><creatorcontrib>Noone, Dylan</creatorcontrib><creatorcontrib>Liu, Xia</creatorcontrib><creatorcontrib>Richardson, Andrew P.</creatorcontrib><creatorcontrib>Chacinska, Agnieszka</creatorcontrib><creatorcontrib>Collinson, Ian</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pereira, Gonçalo C.</au><au>Allen, William J.</au><au>Watkins, Daniel W.</au><au>Buddrus, Lisa</au><au>Noone, Dylan</au><au>Liu, Xia</au><au>Richardson, Andrew P.</au><au>Chacinska, Agnieszka</au><au>Collinson, Ian</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2019-04-05</date><risdate>2019</risdate><volume>431</volume><issue>8</issue><spage>1689</spage><epage>1699</epage><pages>1689-1699</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><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.
[Display omitted]
•Conventional methods for monitoring protein translocation are laborious and discontinuous and lack kinetic detail.•A split NanoLuc system was adapted for real-time monitoring of protein translocation through the bacterial Sec system and the mitochondrial Tim23 complex.•The new assay reduces the timescale of data acquisition from days to minutes.•It produces high-quality results suitable for kinetic fitting and model derivation.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>30878481</pmid><doi>10.1016/j.jmb.2019.03.007</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0001-9638-0615</orcidid><orcidid>https://orcid.org/0000-0003-0524-7021</orcidid><orcidid>https://orcid.org/0000-0002-2832-2568</orcidid><orcidid>https://orcid.org/0000-0003-3825-5036</orcidid><orcidid>https://orcid.org/0000-0002-3931-0503</orcidid><orcidid>https://orcid.org/0000-0002-9513-4786</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of molecular biology, 2019-04, Vol.431 (8), p.1689-1699 |
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| language | eng |
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| source | ScienceDirect Freedom Collection |
| subjects | bacterial Sec system Escherichia coli - metabolism Escherichia coli Infections - microbiology Escherichia coli Proteins - metabolism Humans live assay Luciferases - metabolism Luminescent Agents - metabolism Luminescent Measurements - methods Mitochondrial Membranes - metabolism mitochondrial protein import NanoLuc protein translocation Protein Transport Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - metabolism SEC Translocation Channels - metabolism |
| title | A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes |
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