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Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH
Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoreticall...
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| Published in: | Angewandte Chemie 2020-06, Vol.132 (26), p.10497-10501 |
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| Main Authors: | , , , , , , |
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| container_end_page | 10501 |
| container_issue | 26 |
| container_start_page | 10497 |
| container_title | Angewandte Chemie |
| container_volume | 132 |
| creator | Alik, Ania Bouguechtouli, Chafiaa Julien, Manon Bermel, Wolfgang Ghouil, Rania Zinn‐Justin, Sophie Theillet, Francois‐Xavier |
| description | Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue‐specific level using 1H‐15N NMR spectroscopy, but is often limited by low signal‐to‐noise at pH>7 and T>293 K. We have developed an improved 13Cα‐13CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic‐resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.
An NMR spectroscopy method has been developed to monitor site‐specific phosphorylation at high pH and temperature, both of which are necessary for kinase activity but have previously prevented the thorough characterization of disordered proteins. This approach uses sensitivity‐enhanced 13Cα‐13CO experiments and is demonstrated using Mdm2, BRCA2, and Oct4. Using this approach, many more phosphorylation cases can now beinvestigated using NMR spectroscopy. |
| doi_str_mv | 10.1002/ange.202002288 |
| format | article |
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An NMR spectroscopy method has been developed to monitor site‐specific phosphorylation at high pH and temperature, both of which are necessary for kinase activity but have previously prevented the thorough characterization of disordered proteins. This approach uses sensitivity‐enhanced 13Cα‐13CO experiments and is demonstrated using Mdm2, BRCA2, and Oct4. Using this approach, many more phosphorylation cases can now beinvestigated using NMR spectroscopy.</description><identifier>ISSN: 0044-8249</identifier><identifier>EISSN: 1521-3757</identifier><identifier>DOI: 10.1002/ange.202002288</identifier><language>eng</language><publisher>Weinheim: Wiley Subscription Services, Inc</publisher><subject>BRCA2 protein ; Breast cancer ; cell signaling ; Chemistry ; Deoxyribonucleic acid ; DNA ; DNA repair ; Gene regulation ; Kinases ; Magnetic resonance spectroscopy ; MDM2 protein ; Monitoring ; NMR ; NMR spectroscopy ; Nuclear magnetic resonance ; Oct-4 protein ; pH effects ; Phosphorylation ; Physiology ; protein modifications ; Proteins ; Sensitivity enhancement ; Spectroscopy ; Spectrum analysis ; Temperature</subject><ispartof>Angewandte Chemie, 2020-06, Vol.132 (26), p.10497-10501</ispartof><rights>2020 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><orcidid>0000-0002-3264-210X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,783,787,27936,27937</link.rule.ids></links><search><creatorcontrib>Alik, Ania</creatorcontrib><creatorcontrib>Bouguechtouli, Chafiaa</creatorcontrib><creatorcontrib>Julien, Manon</creatorcontrib><creatorcontrib>Bermel, Wolfgang</creatorcontrib><creatorcontrib>Ghouil, Rania</creatorcontrib><creatorcontrib>Zinn‐Justin, Sophie</creatorcontrib><creatorcontrib>Theillet, Francois‐Xavier</creatorcontrib><title>Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH</title><title>Angewandte Chemie</title><description>Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue‐specific level using 1H‐15N NMR spectroscopy, but is often limited by low signal‐to‐noise at pH>7 and T>293 K. We have developed an improved 13Cα‐13CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic‐resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.
An NMR spectroscopy method has been developed to monitor site‐specific phosphorylation at high pH and temperature, both of which are necessary for kinase activity but have previously prevented the thorough characterization of disordered proteins. This approach uses sensitivity‐enhanced 13Cα‐13CO experiments and is demonstrated using Mdm2, BRCA2, and Oct4. Using this approach, many more phosphorylation cases can now beinvestigated using NMR spectroscopy.</description><subject>BRCA2 protein</subject><subject>Breast cancer</subject><subject>cell signaling</subject><subject>Chemistry</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA repair</subject><subject>Gene regulation</subject><subject>Kinases</subject><subject>Magnetic resonance spectroscopy</subject><subject>MDM2 protein</subject><subject>Monitoring</subject><subject>NMR</subject><subject>NMR spectroscopy</subject><subject>Nuclear magnetic resonance</subject><subject>Oct-4 protein</subject><subject>pH effects</subject><subject>Phosphorylation</subject><subject>Physiology</subject><subject>protein modifications</subject><subject>Proteins</subject><subject>Sensitivity enhancement</subject><subject>Spectroscopy</subject><subject>Spectrum analysis</subject><subject>Temperature</subject><issn>0044-8249</issn><issn>1521-3757</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><recordid>eNo9kMFOAjEQhhujiYhePTfxvNh2W7Z7JATBBNAI96YuLZQsbe12NevJR_AZfRJLNJxmvsk_M8kHwC1GA4wQuZd2qwYEkdQTzs9ADzOCs7xgxTnoIURpxgktL8FV0-wRQkNSlD3wuVK2MdG8m9j9fH1P7E7aSm0gzscJl4sXuPKqisE1lfMd1C7AhbMmumDsFi7aOpq0ruDzzjV-50JXy2ichTKmUdcYV7utqWQN1-rgVZCxDQpKu4F-dg0utKwbdfNf-2D9MFmPZ9n8afo4Hs0zX3CeaU2J1q9YKqaHrMIaa024LDlFWDPCMOaFLipNlGQ55Zon3tBcorzUBePDvA_u_s764N5a1USxd22w6aMgFCcJDJc0pcq_1IepVSd8MAcZOoGROLoVR7fi5FaMltPJifJf519zww</recordid><startdate>20200622</startdate><enddate>20200622</enddate><creator>Alik, Ania</creator><creator>Bouguechtouli, Chafiaa</creator><creator>Julien, Manon</creator><creator>Bermel, Wolfgang</creator><creator>Ghouil, Rania</creator><creator>Zinn‐Justin, Sophie</creator><creator>Theillet, Francois‐Xavier</creator><general>Wiley Subscription Services, Inc</general><scope>7SR</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>JG9</scope><scope>L7M</scope><orcidid>https://orcid.org/0000-0002-3264-210X</orcidid></search><sort><creationdate>20200622</creationdate><title>Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH</title><author>Alik, Ania ; Bouguechtouli, Chafiaa ; Julien, Manon ; Bermel, Wolfgang ; Ghouil, Rania ; Zinn‐Justin, Sophie ; Theillet, Francois‐Xavier</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p788-ff42ffb1ae5f65c1f1ff28a98401f5251187f7cf2ea5348f8187d43a039f75863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>BRCA2 protein</topic><topic>Breast cancer</topic><topic>cell signaling</topic><topic>Chemistry</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA repair</topic><topic>Gene regulation</topic><topic>Kinases</topic><topic>Magnetic resonance spectroscopy</topic><topic>MDM2 protein</topic><topic>Monitoring</topic><topic>NMR</topic><topic>NMR spectroscopy</topic><topic>Nuclear magnetic resonance</topic><topic>Oct-4 protein</topic><topic>pH effects</topic><topic>Phosphorylation</topic><topic>Physiology</topic><topic>protein modifications</topic><topic>Proteins</topic><topic>Sensitivity enhancement</topic><topic>Spectroscopy</topic><topic>Spectrum analysis</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Alik, Ania</creatorcontrib><creatorcontrib>Bouguechtouli, Chafiaa</creatorcontrib><creatorcontrib>Julien, Manon</creatorcontrib><creatorcontrib>Bermel, Wolfgang</creatorcontrib><creatorcontrib>Ghouil, Rania</creatorcontrib><creatorcontrib>Zinn‐Justin, Sophie</creatorcontrib><creatorcontrib>Theillet, Francois‐Xavier</creatorcontrib><collection>Engineered Materials Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Materials Research Database</collection><collection>Advanced Technologies Database with Aerospace</collection><jtitle>Angewandte Chemie</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Alik, Ania</au><au>Bouguechtouli, Chafiaa</au><au>Julien, Manon</au><au>Bermel, Wolfgang</au><au>Ghouil, Rania</au><au>Zinn‐Justin, Sophie</au><au>Theillet, Francois‐Xavier</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH</atitle><jtitle>Angewandte Chemie</jtitle><date>2020-06-22</date><risdate>2020</risdate><volume>132</volume><issue>26</issue><spage>10497</spage><epage>10501</epage><pages>10497-10501</pages><issn>0044-8249</issn><eissn>1521-3757</eissn><abstract>Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue‐specific level using 1H‐15N NMR spectroscopy, but is often limited by low signal‐to‐noise at pH>7 and T>293 K. We have developed an improved 13Cα‐13CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic‐resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.
An NMR spectroscopy method has been developed to monitor site‐specific phosphorylation at high pH and temperature, both of which are necessary for kinase activity but have previously prevented the thorough characterization of disordered proteins. This approach uses sensitivity‐enhanced 13Cα‐13CO experiments and is demonstrated using Mdm2, BRCA2, and Oct4. Using this approach, many more phosphorylation cases can now beinvestigated using NMR spectroscopy.</abstract><cop>Weinheim</cop><pub>Wiley Subscription Services, Inc</pub><doi>10.1002/ange.202002288</doi><tpages>5</tpages><orcidid>https://orcid.org/0000-0002-3264-210X</orcidid></addata></record> |
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| language | eng |
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| source | Wiley-Blackwell Read & Publish Collection |
| subjects | BRCA2 protein Breast cancer cell signaling Chemistry Deoxyribonucleic acid DNA DNA repair Gene regulation Kinases Magnetic resonance spectroscopy MDM2 protein Monitoring NMR NMR spectroscopy Nuclear magnetic resonance Oct-4 protein pH effects Phosphorylation Physiology protein modifications Proteins Sensitivity enhancement Spectroscopy Spectrum analysis Temperature |
| title | Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH |
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