Methyl Groups as Probes for Proteins and Complexes in In-Cell NMR Experiments
Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which posses...
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| Veröffentlicht in: | Journal of the American Chemical Society 2004-06, Vol.126 (22), p.7119-7125 |
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| container_end_page | 7125 |
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| container_issue | 22 |
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| container_title | Journal of the American Chemical Society |
| container_volume | 126 |
| creator | Serber, Zach Straub, Wesley Corsini, Lorenzo Nomura, Anson M Shimba, Nobuhisa Craik, Charles S Ortiz de Montellano, Paul Dötsch, Volker |
| description | Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [13C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs. |
| doi_str_mv | 10.1021/ja049977k |
| format | Article |
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We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [13C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.</description><identifier>ISSN: 0002-7863</identifier><identifier>EISSN: 1520-5126</identifier><identifier>DOI: 10.1021/ja049977k</identifier><identifier>PMID: 15174883</identifier><identifier>CODEN: JACSAT</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Carbon Isotopes - chemistry ; Cells - chemistry ; Escherichia coli - chemistry ; Fundamental and applied biological sciences. 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Am. Chem. Soc</addtitle><description>Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [13C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Carbon Isotopes - chemistry</subject><subject>Cells - chemistry</subject><subject>Escherichia coli - chemistry</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>General aspects, investigation methods</subject><subject>Methionine - chemistry</subject><subject>Methylation</subject><subject>Nuclear Magnetic Resonance, Biomolecular - methods</subject><subject>Pharmaceutical Preparations - chemistry</subject><subject>Pharmaceutical Preparations - metabolism</subject><subject>Proteins</subject><subject>Proteins - chemistry</subject><subject>Pyruvic Acid - chemistry</subject><subject>Tacrolimus Binding Proteins - chemistry</subject><issn>0002-7863</issn><issn>1520-5126</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0MtKxDAUBuAgio6XhS8g3Si4qCZprksdRkdxVLysQ9qeYMdOW5MWxrc3MoO6cJXL-Tj8_AgdEnxGMCXnc4uZ1lK-b6AR4RSnnFCxiUYYY5pKJbIdtBvCPD4ZVWQb7RBOJFMqG6HZDPq3zzq59u3QhcSG5NG3OYTEtf772kPVxO-mTMbtoqthGUdVk9w06RjqOrmfPSWTZQe-WkDTh3205Wwd4GB97qHXq8nLeJrePVzfjC_uUssY71ORW6GZ06LMqSMKS21JKYgFxcDlipc5z0pXyJzzjJWKcwqOa1ZAXhZCUZrtoZPV3s63HwOE3iyqUMRAtoF2CEYSrRkhOsLTFSx8G4IHZ7oY1fpPQ7D57s78dBft0XrpkC-g_JXrsiI4XgMbCls7b5uiCn-cyiQXLLp05arQw_Jnbv27ETIS8_L4bGZTfHvJplfm6XevLYKZt4NvYnf_BPwC6DWQ3Q</recordid><startdate>20040609</startdate><enddate>20040609</enddate><creator>Serber, Zach</creator><creator>Straub, Wesley</creator><creator>Corsini, Lorenzo</creator><creator>Nomura, Anson M</creator><creator>Shimba, Nobuhisa</creator><creator>Craik, Charles S</creator><creator>Ortiz de Montellano, Paul</creator><creator>Dötsch, Volker</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>IQODW</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></search><sort><creationdate>20040609</creationdate><title>Methyl Groups as Probes for Proteins and Complexes in In-Cell NMR Experiments</title><author>Serber, Zach ; Straub, Wesley ; Corsini, Lorenzo ; Nomura, Anson M ; Shimba, Nobuhisa ; Craik, Charles S ; Ortiz de Montellano, Paul ; Dötsch, Volker</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a445t-6ba694f96db2f18079a1d61ae84efb85db53dfc7b5534d8552ef594cebdc68223</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Carbon Isotopes - chemistry</topic><topic>Cells - chemistry</topic><topic>Escherichia coli - chemistry</topic><topic>Fundamental and applied biological sciences. 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| ispartof | Journal of the American Chemical Society, 2004-06, Vol.126 (22), p.7119-7125 |
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| source | ACS Publications; MEDLINE |
| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Carbon Isotopes - chemistry Cells - chemistry Escherichia coli - chemistry Fundamental and applied biological sciences. Psychology General aspects, investigation methods Methionine - chemistry Methylation Nuclear Magnetic Resonance, Biomolecular - methods Pharmaceutical Preparations - chemistry Pharmaceutical Preparations - metabolism Proteins Proteins - chemistry Pyruvic Acid - chemistry Tacrolimus Binding Proteins - chemistry |
| title | Methyl Groups as Probes for Proteins and Complexes in In-Cell NMR Experiments |
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