Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments
Identifieur interne : 000238 ( Pmc/Corpus ); précédent : 000237; suivant : 000239Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments
Auteurs : Koh Takeuchi ; Gregory Heffron ; Zhen-Yu J. Sun ; Dominique P. Frueh ; Gerhard WagnerSource :
- Journal of biomolecular NMR [ 0925-2738 ] ; 2010.
Abstract
Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as 13C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel 15N direct-detection experiments. The CAN experiment sequentially connects amide 15N resonances using 13Cα chemical shift matching, and the CON experiment connects the preceding 13C′ nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding 13C-detecting experiment, NCA-DIPAP or NCO-IPAP (
Url:
DOI: 10.1007/s10858-010-9430-z
PubMed: 20556482
PubMed Central: 2946331
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PMC:2946331Le document en format XML
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<author><name sortKey="Takeuchi, Koh" sort="Takeuchi, Koh" uniqKey="Takeuchi K" first="Koh" last="Takeuchi">Koh Takeuchi</name>
</author>
<author><name sortKey="Heffron, Gregory" sort="Heffron, Gregory" uniqKey="Heffron G" first="Gregory" last="Heffron">Gregory Heffron</name>
</author>
<author><name sortKey="Sun, Zhen Yu J" sort="Sun, Zhen Yu J" uniqKey="Sun Z" first="Zhen-Yu J." last="Sun">Zhen-Yu J. Sun</name>
</author>
<author><name sortKey="Frueh, Dominique P" sort="Frueh, Dominique P" uniqKey="Frueh D" first="Dominique P." last="Frueh">Dominique P. Frueh</name>
</author>
<author><name sortKey="Wagner, Gerhard" sort="Wagner, Gerhard" uniqKey="Wagner G" first="Gerhard" last="Wagner">Gerhard Wagner</name>
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<author><name sortKey="Takeuchi, Koh" sort="Takeuchi, Koh" uniqKey="Takeuchi K" first="Koh" last="Takeuchi">Koh Takeuchi</name>
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<author><name sortKey="Heffron, Gregory" sort="Heffron, Gregory" uniqKey="Heffron G" first="Gregory" last="Heffron">Gregory Heffron</name>
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<author><name sortKey="Sun, Zhen Yu J" sort="Sun, Zhen Yu J" uniqKey="Sun Z" first="Zhen-Yu J." last="Sun">Zhen-Yu J. Sun</name>
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<author><name sortKey="Frueh, Dominique P" sort="Frueh, Dominique P" uniqKey="Frueh D" first="Dominique P." last="Frueh">Dominique P. Frueh</name>
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<author><name sortKey="Wagner, Gerhard" sort="Wagner, Gerhard" uniqKey="Wagner G" first="Gerhard" last="Wagner">Gerhard Wagner</name>
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<series><title level="j">Journal of biomolecular NMR</title>
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<front><div type="abstract" xml:lang="en"><p id="P1">Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as <sup>13</sup>
C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel <sup>15</sup>
N direct-detection experiments. The CAN experiment sequentially connects amide <sup>15</sup>
N resonances using <sup>13</sup>
C<sup>α</sup>
chemical shift matching, and the CON experiment connects the preceding <sup>13</sup>
C′ nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding <sup>13</sup>
C-detecting experiment, NCA-DIPAP or NCO-IPAP (<xref ref-type="bibr" rid="R5">Bermel et al. 2006b</xref>
; <xref ref-type="bibr" rid="R35">Takeuchi et al. 2008</xref>
). However, the disadvantage due to the lower γ is counteracted by the slower <sup>15</sup>
N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the <sup>15</sup>
N observe CAN experiment is 16% higher than in the <sup>13</sup>
C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field C<sup>α</sup>
resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D<sub>2</sub>
O. Thus, these features and the superior resolution of <sup>15</sup>
N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.</p>
</div>
</front>
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<pmc article-type="research-article" xml:lang="EN"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">9110829</journal-id>
<journal-id journal-id-type="pubmed-jr-id">2209</journal-id>
<journal-id journal-id-type="nlm-ta">J Biomol NMR</journal-id>
<journal-title>Journal of biomolecular NMR</journal-title>
<issn pub-type="ppub">0925-2738</issn>
<issn pub-type="epub">1573-5001</issn>
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<article-meta><article-id pub-id-type="pmid">20556482</article-id>
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<article-id pub-id-type="doi">10.1007/s10858-010-9430-z</article-id>
<article-id pub-id-type="manuscript">NIHMS223248</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
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<title-group><article-title>Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Takeuchi</surname>
<given-names>Koh</given-names>
</name>
<aff id="A1">Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA</aff>
<aff id="A2">Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan</aff>
</contrib>
<contrib contrib-type="author"><name><surname>Heffron</surname>
<given-names>Gregory</given-names>
</name>
<aff id="A3">Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA</aff>
</contrib>
<contrib contrib-type="author"><name><surname>Sun</surname>
<given-names>Zhen-Yu J.</given-names>
</name>
<aff id="A4">Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA</aff>
</contrib>
<contrib contrib-type="author"><name><surname>Frueh</surname>
<given-names>Dominique P.</given-names>
</name>
<aff id="A5">Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA</aff>
<aff id="A6">Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA</aff>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Wagner</surname>
<given-names>Gerhard</given-names>
</name>
<email>gerhard-wagner@hms.harvard.edu</email>
<aff id="A7">Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA</aff>
</contrib>
</contrib-group>
<pub-date pub-type="nihms-submitted"><day>8</day>
<month>9</month>
<year>2010</year>
</pub-date>
<pub-date pub-type="epub"><day>17</day>
<month>6</month>
<year>2010</year>
</pub-date>
<pub-date pub-type="ppub"><month>8</month>
<year>2010</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>1</day>
<month>8</month>
<year>2011</year>
</pub-date>
<volume>47</volume>
<issue>4</issue>
<fpage>271</fpage>
<lpage>282</lpage>
<permissions><copyright-statement>© Springer Science+Business Media B.V. 2010</copyright-statement>
<copyright-year>2010</copyright-year>
</permissions>
<abstract><p id="P1">Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as <sup>13</sup>
C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel <sup>15</sup>
N direct-detection experiments. The CAN experiment sequentially connects amide <sup>15</sup>
N resonances using <sup>13</sup>
C<sup>α</sup>
chemical shift matching, and the CON experiment connects the preceding <sup>13</sup>
C′ nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding <sup>13</sup>
C-detecting experiment, NCA-DIPAP or NCO-IPAP (<xref ref-type="bibr" rid="R5">Bermel et al. 2006b</xref>
; <xref ref-type="bibr" rid="R35">Takeuchi et al. 2008</xref>
). However, the disadvantage due to the lower γ is counteracted by the slower <sup>15</sup>
N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the <sup>15</sup>
N observe CAN experiment is 16% higher than in the <sup>13</sup>
C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field C<sup>α</sup>
resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D<sub>2</sub>
O. Thus, these features and the superior resolution of <sup>15</sup>
N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.</p>
</abstract>
<kwd-group><kwd><sup>15</sup>
N direct detection</kwd>
<kwd>CAN</kwd>
<kwd>CON</kwd>
<kwd>HSQC</kwd>
<kwd>Nuclear magnetic resonance (NMR)</kwd>
<kwd>Resonance assignment</kwd>
</kwd-group>
<contract-num rid="AI1">R01 AI037581-14
||AI</contract-num>
<contract-num rid="GM1">P01 GM047467-20
||GM</contract-num>
<contract-num rid="GM1">P01 GM047467-199002
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||GM</contract-num>
<contract-num rid="GM1">P01 GM047467-189001
||GM</contract-num>
<contract-sponsor id="AI1">National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID</contract-sponsor>
<contract-sponsor id="GM1">National Institute of General Medical Sciences : NIGMS</contract-sponsor>
</article-meta>
</front>
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