Solid-State NMR Structure Determination from Diagonal-Compensated, Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints
Identifieur interne : 000691 ( Pmc/Corpus ); précédent : 000690; suivant : 000692Solid-State NMR Structure Determination from Diagonal-Compensated, Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints
Auteurs : Rasmus Linser ; Benjamin Bardiaux ; Loren X0a B. Andreas ; Sven G. Hyberts ; Vanessa K. Morris ; Guido Pintacuda ; Margaret Sunde ; Ann H. Kwan ; Gerhard WagnerSource :
- Journal of the American Chemical Society [ 0002-7863 ] ; 2014.
Abstract
We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and 1H back-exchanged proteins. To achieve this, we use homonuclear 1H–1H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.
Url:
DOI: 10.1021/ja504603g
PubMed: 24988008
PubMed Central: 4132958
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PMC:4132958Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Solid-State
NMR Structure Determination from Diagonal-Compensated,
Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints</title><author><name sortKey="Linser, Rasmus" sort="Linser, Rasmus" uniqKey="Linser R" first="Rasmus" last="Linser">Rasmus Linser</name><affiliation><nlm:aff id="aff6"><institution>Max-Planck Institute for Biophysical Chemistry</institution>, Am Fassberg 11, 37077 Göttingen,<country>Germany</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">School of Chemistry,<institution>University of New South Wales</institution>, Sydney NSW 2052,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Bardiaux, Benjamin" sort="Bardiaux, Benjamin" uniqKey="Bardiaux B" first="Benjamin" last="Bardiaux">Benjamin Bardiaux</name><affiliation><nlm:aff id="aff3">Unité de Bioinformatique Structurale, CNRS UMR 3528,<institution>Institut Pasteur</institution>, Paris CEDEX 15,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Andreas, Loren X0a B" sort="Andreas, Loren X0a B" uniqKey="Andreas L" first="Loren X0a B." last="Andreas">Loren X0a B. Andreas</name><affiliation><nlm:aff id="aff4"><institution>Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/Université de Lyon 1</institution>, 69100 Villeurbanne,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Hyberts, Sven G" sort="Hyberts, Sven G" uniqKey="Hyberts S" first="Sven G." last="Hyberts">Sven G. Hyberts</name><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Morris, Vanessa K" sort="Morris, Vanessa K" uniqKey="Morris V" first="Vanessa K." last="Morris">Vanessa K. Morris</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Pintacuda, Guido" sort="Pintacuda, Guido" uniqKey="Pintacuda G" first="Guido" last="Pintacuda">Guido Pintacuda</name><affiliation><nlm:aff id="aff4"><institution>Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/Université de Lyon 1</institution>, 69100 Villeurbanne,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Sunde, Margaret" sort="Sunde, Margaret" uniqKey="Sunde M" first="Margaret" last="Sunde">Margaret Sunde</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Kwan, Ann H" sort="Kwan, Ann H" uniqKey="Kwan A" first="Ann H." last="Kwan">Ann H. Kwan</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Wagner, Gerhard" sort="Wagner, Gerhard" uniqKey="Wagner G" first="Gerhard" last="Wagner">Gerhard Wagner</name><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24988008</idno><idno type="pmc">4132958</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132958</idno><idno type="RBID">PMC:4132958</idno><idno type="doi">10.1021/ja504603g</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000691</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000691</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Solid-State
NMR Structure Determination from Diagonal-Compensated,
Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints</title><author><name sortKey="Linser, Rasmus" sort="Linser, Rasmus" uniqKey="Linser R" first="Rasmus" last="Linser">Rasmus Linser</name><affiliation><nlm:aff id="aff6"><institution>Max-Planck Institute for Biophysical Chemistry</institution>, Am Fassberg 11, 37077 Göttingen,<country>Germany</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">School of Chemistry,<institution>University of New South Wales</institution>, Sydney NSW 2052,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Bardiaux, Benjamin" sort="Bardiaux, Benjamin" uniqKey="Bardiaux B" first="Benjamin" last="Bardiaux">Benjamin Bardiaux</name><affiliation><nlm:aff id="aff3">Unité de Bioinformatique Structurale, CNRS UMR 3528,<institution>Institut Pasteur</institution>, Paris CEDEX 15,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Andreas, Loren X0a B" sort="Andreas, Loren X0a B" uniqKey="Andreas L" first="Loren X0a B." last="Andreas">Loren X0a B. Andreas</name><affiliation><nlm:aff id="aff4"><institution>Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/Université de Lyon 1</institution>, 69100 Villeurbanne,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Hyberts, Sven G" sort="Hyberts, Sven G" uniqKey="Hyberts S" first="Sven G." last="Hyberts">Sven G. Hyberts</name><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Morris, Vanessa K" sort="Morris, Vanessa K" uniqKey="Morris V" first="Vanessa K." last="Morris">Vanessa K. Morris</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Pintacuda, Guido" sort="Pintacuda, Guido" uniqKey="Pintacuda G" first="Guido" last="Pintacuda">Guido Pintacuda</name><affiliation><nlm:aff id="aff4"><institution>Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/Université de Lyon 1</institution>, 69100 Villeurbanne,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Sunde, Margaret" sort="Sunde, Margaret" uniqKey="Sunde M" first="Margaret" last="Sunde">Margaret Sunde</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Kwan, Ann H" sort="Kwan, Ann H" uniqKey="Kwan A" first="Ann H." last="Kwan">Ann H. Kwan</name><affiliation><nlm:aff id="aff5">School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Wagner, Gerhard" sort="Wagner, Gerhard" uniqKey="Wagner G" first="Gerhard" last="Wagner">Gerhard Wagner</name><affiliation><nlm:aff id="aff1">Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of the American Chemical Society</title><idno type="ISSN">0002-7863</idno><idno type="eISSN">1520-5126</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ja-2014-04603g_0006" id="ab-tgr1"></graphic></p><p>We report acquisition of diagonal-compensated
protein structural
restraints from four-dimensional solid-state NMR spectra on extensively
deuterated and <sup>1</sup>H back-exchanged proteins. To achieve this,
we use homonuclear <sup>1</sup>H–<sup>1</sup>H correlations
with diagonal suppression and nonuniform sampling (NUS). Suppression
of the diagonal allows the accurate identification of cross-peaks
which are otherwise obscured by the strong autocorrelation or whose
intensity is biased due to partial overlap with the diagonal. The
approach results in unambiguous spectral interpretation and relatively
few but reliable restraints for structure calculation. In addition,
the diagonal suppression produces a spectrum with low dynamic range
for which ultrasparse NUS data sets can be readily reconstructed,
allowing straightforward application of NUS with only 2% sampling
density with the advantage of more heavily sampling time-domain regions
of high signal intensity. The method is demonstrated here for two
proteins, α-spectrin SH3 microcrystals and hydrophobin functional
amyloids. For the case of SH3, suppression of the diagonal results
in facilitated identification of unambiguous restraints and improvement
of the quality of the calculated structural ensemble compared to nondiagonal-suppressed
4D spectra. For the only partly assigned hydrophobin rodlets, the
structure is yet unknown. Applied to this protein of biological significance
with large inhomogeneous broadening, the method allows identification
of unambiguous crosspeaks that are otherwise obscured by the diagonal.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Cady, S D" uniqKey="Cady S">S. D. Cady</name></author><author><name sortKey="Schmidt Rohr, K" uniqKey="Schmidt Rohr K">K. Schmidt-Rohr</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J. Wang</name></author><author><name sortKey="Soto, C" uniqKey="Soto C">C. Soto</name></author><author><name sortKey="Degrado, W F" uniqKey="Degrado W">W. F. DeGrado</name></author><author><name sortKey="Hong, M" uniqKey="Hong M">M. Hong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Loquet, A" uniqKey="Loquet A">A. Loquet</name></author><author><name sortKey="Sgourakis, N G" uniqKey="Sgourakis N">N. G. Sgourakis</name></author><author><name sortKey="Gupta, R" uniqKey="Gupta R">R. Gupta</name></author><author><name sortKey="Giller, K" uniqKey="Giller K">K. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Am Chem Soc</journal-id><journal-id journal-id-type="iso-abbrev">J. Am. Chem. Soc</journal-id><journal-id journal-id-type="publisher-id">ja</journal-id><journal-id journal-id-type="coden">jacsat</journal-id><journal-title-group><journal-title>Journal of the American Chemical Society</journal-title></journal-title-group><issn pub-type="ppub">0002-7863</issn><issn pub-type="epub">1520-5126</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24988008</article-id><article-id pub-id-type="pmc">4132958</article-id><article-id pub-id-type="doi">10.1021/ja504603g</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Solid-State
NMR Structure Determination from Diagonal-Compensated,
Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="ath1"><name><surname>Linser</surname><given-names>Rasmus</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff6" ref-type="aff">¶</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Bardiaux</surname><given-names>Benjamin</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Andreas</surname><given-names>Loren
B.</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Hyberts</surname><given-names>Sven G.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Morris</surname><given-names>Vanessa K.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref><xref rid="notes-1" ref-type="notes">#</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Pintacuda</surname><given-names>Guido</given-names></name><xref rid="aff4" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Sunde</surname><given-names>Margaret</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>Kwan</surname><given-names>Ann H.</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Wagner</surname><given-names>Gerhard</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><aff id="aff6"><label>¶</label><institution>Max-Planck Institute for Biophysical Chemistry</institution>, Am Fassberg 11, 37077 Göttingen,<country>Germany</country></aff><aff id="aff1"><label>†</label>Department of Biological Chemistry and Molecular Pharmacology,<institution>Harvard Medical School</institution>, Boston, Massachusetts 02115,<country>United States</country></aff><aff id="aff2"><label>‡</label>School of Chemistry,<institution>University of New South Wales</institution>, Sydney NSW 2052,<country>Australia</country></aff><aff id="aff3"><label>§</label>Unité de Bioinformatique Structurale, CNRS UMR 3528,<institution>Institut Pasteur</institution>, Paris CEDEX 15,<country>France</country></aff><aff id="aff4"><label>∥</label><institution>Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon/Université de Lyon 1</institution>, 69100 Villeurbanne,<country>France</country></aff><aff id="aff5"><label>⊥</label>School of Medical Sciences and School of Molecular Bioscience,<institution>University of Sydney</institution>, Sydney NSW 2006,<country>Australia</country></aff></contrib-group><author-notes><corresp id="cor1"><email>rasmus.linser@gmx.de</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>02</day><month>07</month><year>2015</year></pub-date><pub-date pub-type="epub"><day>02</day><month>07</month><year>2014</year></pub-date><pub-date pub-type="ppub"><day>06</day><month>08</month><year>2014</year></pub-date><volume>136</volume><issue>31</issue><fpage>11002</fpage><lpage>11010</lpage><history><date date-type="received"><day>08</day><month>05</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 American Chemical
Society</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>American Chemical
Society</copyright-holder><license><license-p><ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_termsofuse.html">Terms of Use</ext-link></license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="ja-2014-04603g_0006" id="ab-tgr1"></graphic></p><p>We report acquisition of diagonal-compensated
protein structural
restraints from four-dimensional solid-state NMR spectra on extensively
deuterated and <sup>1</sup>H back-exchanged proteins. To achieve this,
we use homonuclear <sup>1</sup>H–<sup>1</sup>H correlations
with diagonal suppression and nonuniform sampling (NUS). Suppression
of the diagonal allows the accurate identification of cross-peaks
which are otherwise obscured by the strong autocorrelation or whose
intensity is biased due to partial overlap with the diagonal. The
approach results in unambiguous spectral interpretation and relatively
few but reliable restraints for structure calculation. In addition,
the diagonal suppression produces a spectrum with low dynamic range
for which ultrasparse NUS data sets can be readily reconstructed,
allowing straightforward application of NUS with only 2% sampling
density with the advantage of more heavily sampling time-domain regions
of high signal intensity. The method is demonstrated here for two
proteins, α-spectrin SH3 microcrystals and hydrophobin functional
amyloids. For the case of SH3, suppression of the diagonal results
in facilitated identification of unambiguous restraints and improvement
of the quality of the calculated structural ensemble compared to nondiagonal-suppressed
4D spectra. For the only partly assigned hydrophobin rodlets, the
structure is yet unknown. Applied to this protein of biological significance
with large inhomogeneous broadening, the method allows identification
of unambiguous crosspeaks that are otherwise obscured by the diagonal.</p></abstract><funding-group><funding-statement><funding-source>National Institutes of Health, United States</funding-source></funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>ja504603g</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>ja-2014-04603g</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="sec1"><title>Introduction</title><p>In recent years, solid-state
NMR has evolved as a general tool
to probe the structure and dynamics of biological macromolecules<sup><xref ref-type="bibr" rid="ref1">1</xref>−<xref ref-type="bibr" rid="ref5">5</xref></sup> and a wide range of other materials.<sup><xref ref-type="bibr" rid="ref6">6</xref>−<xref ref-type="bibr" rid="ref11">11</xref></sup> The technique has found particular applicability in the investigation
of solid systems that have a partial intrinsic disorder, such as fibrillar
proteins<sup><xref ref-type="bibr" rid="ref12">12</xref>−<xref ref-type="bibr" rid="ref15">15</xref></sup> or membrane proteins,<sup><xref ref-type="bibr" rid="ref3">3</xref>,<xref ref-type="bibr" rid="ref16">16</xref>−<xref ref-type="bibr" rid="ref21">21</xref></sup> which are embedded in a dynamic environment or display mobility
themselves. Structural assessment by NMR is to a great extent based
on the availability of individual distances between protein spin pairs.
Existing methods have demonstrated highly accurate structures mostly
based on a high number of crosspeaks per residue in few spectral dimensions.<sup><xref ref-type="bibr" rid="ref22">22</xref>−<xref ref-type="bibr" rid="ref25">25</xref></sup> The usefulness of spatial correlations, however, depends on reliable
assignments and sufficient spectral resolution.</p><p>When applied
to proteins with many resonances or for samples with
structural heterogeneity and/or dynamic behavior, substantial peak
overlap is encountered. To reduce the probability of shift degeneracy,
recent efforts in solid-state NMR have been directed toward extending
the commonly encountered <sup>13</sup>C and <sup>15</sup>N spectral
dimensions to additional nuclei, particularly <sup>1</sup>H. The accessibility
of proton chemical shifts by fast magic angle spinning (fast MAS)
and tailored deuteration approaches<sup><xref ref-type="bibr" rid="ref26">26</xref>−<xref ref-type="bibr" rid="ref30">30</xref></sup> has greatly facilitated powerful <sup>1</sup>H/<sup>15</sup>N/<sup>13</sup>C triple-resonance approaches recently.<sup><xref ref-type="bibr" rid="ref31">31</xref>−<xref ref-type="bibr" rid="ref34">34</xref></sup> Techniques currently developed for spinning frequencies of up to
100 kHz and higher allow narrow proton line widths even in the absence
of deuteration.<sup><xref ref-type="bibr" rid="ref35">35</xref>−<xref ref-type="bibr" rid="ref37">37</xref></sup> Proton detection has so far been successful in providing
improved resolution and reliability in a new generation of solid-state
NMR experiments mainly for resonance assignment and characterization
of dynamics.</p><p>The most important approach for measuring distance
restraints and
other distance-related information to enable structure calculation
relies on magnetization transfers between spins in spatial proximity.
Commonly applied methods using fully protonated samples measure <sup>13</sup>C–<sup>13</sup>C or <sup>13</sup>C–<sup>15</sup>N contacts,<sup><xref ref-type="bibr" rid="ref38">38</xref>−<xref ref-type="bibr" rid="ref42">42</xref></sup> often enabled by exploitation of the proton-dipolar-coupling network,
and yield large quantities of close and predominantly intraresidual
contacts.<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref25">25</xref></sup> Extensive deuteration approaches yield a
reduced set of restraints,<sup><xref ref-type="bibr" rid="ref43">43</xref>,<xref ref-type="bibr" rid="ref44">44</xref></sup> but these exclusively
connect amide and/or methyl protons over relatively long distances
and with high resolution. Also, they are little affected by spin diffusion
and dipolar truncation. Accordingly, these correlations have the potential
to provide accurate and nonredundant restraints for unambiguously
identifiable pairs of residues.</p><p>Recently, excellent methyl–methyl
correlations have been
recorded with solid-state NMR approaching spectral qualities of solution
NMR on samples with stoichiometrically protonated methyl groups (CD<sub>2</sub>H).<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> This is facilitated by the
fast methyl rotations and is achievable even at intermediate MAS frequencies.<sup><xref ref-type="bibr" rid="ref45">45</xref></sup> For the more comprehensive set of distances
between amides, <sup>1</sup>H<sup>N</sup>-back-substituted preparations
have been used at intermediate MAS frequencies of 10–40 kHz.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref44">44</xref>,<xref ref-type="bibr" rid="ref46">46</xref></sup> A partial back-exchange has the
advantage of reducing spin diffusion, indirect transfer processes,
and dipolar truncation effects<sup><xref ref-type="bibr" rid="ref26">26</xref>,<xref ref-type="bibr" rid="ref32">32</xref>,<xref ref-type="bibr" rid="ref46">46</xref>,<xref ref-type="bibr" rid="ref47">47</xref></sup> but comes at the price of larger
sample volumes required. For very fast MAS above 60 kHz, fully <sup>1</sup>H-back-exchanged proteins and lower sample volumes are used
to achieve the highest possible signal-to-noise.<sup><xref ref-type="bibr" rid="ref29">29</xref>,<xref ref-type="bibr" rid="ref34">34</xref></sup> In both cases, however, single-quantum single-quantum (SQ-SQ) correlations
can lead to compromised spectra due to large autocorrelation peaks.
The difficulty is analogous to the case encountered in solution-state
NOESY data.</p><p>Several different approaches have been dedicated
to overcome problems
associated with an intense diagonal signal.<sup><xref ref-type="bibr" rid="ref48">48</xref>−<xref ref-type="bibr" rid="ref55">55</xref></sup> In solution NMR, interleaved experiments for subtraction<sup><xref ref-type="bibr" rid="ref49">49</xref>,<xref ref-type="bibr" rid="ref50">50</xref></sup> as well as spin-state-selective variants based on the ST2 polarization-transfer
scheme have been used.<sup><xref ref-type="bibr" rid="ref51">51</xref>−<xref ref-type="bibr" rid="ref53">53</xref></sup> Both methods sacrifice on the order of 50% of the
sensitivity. Elegant pulse schemes have been developed for solid-state
NMR that result in scaling of signal intensity dependent upon the
distance from the diagonal.<sup><xref ref-type="bibr" rid="ref54">54</xref>,<xref ref-type="bibr" rid="ref55">55</xref></sup> In principle, DQ-filtered
recoupling, using schemes like DREAM<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> or
Post-C7,<sup><xref ref-type="bibr" rid="ref56">56</xref></sup> can obviate the need for diagonal
suppression. (See a review by Demers et al.<sup><xref ref-type="bibr" rid="ref57">57</xref></sup> for a comparison of different recoupling schemes at fast MAS.)</p><p>Diagonal peaks bear little structural information and can potentially
be up to orders of magnitude stronger than the correlations of interest.
The presence of this intense autocorrelation is currently a significant
hindrance to obtaining quality <sup>1</sup>H–<sup>1</sup>H
structural restraints near the diagonal. An easily implementable cancellation
of the diagonal is presented here, which is expected to be especially
desirable also for overcoming the following problems: First, particular
diagonal-related difficulties are expected for future structure calculation
from sparse aliphatic protonation, which now enables straightforward
access to full sets of sharp side-chain proton resonances.<sup><xref ref-type="bibr" rid="ref28">28</xref></sup> The extensive dilution of the aliphatic protons
necessary for this effect will go in hand with extremely large diagonal-to-cross-peak
ratios. Second, evolution times in higher dimensional spectra are
typically truncated even with nonuniform sampling. This results in
severe artifacts from strong peaks, which are also difficult to recognize
in higher dimensional space. In contrast to their overall feasibility
for spectral resolution, higher dimensional spectra are thus particularly
affected by interfering diagonal signals and their artifacts. Third,
α-helical proteins and inhomogeneous samples represent additional
challenges in the presence of intense diagonal signal due to little
spectral amide peak resolution.</p><p>A powerful current method for
encoding high-quality and large-quantity
nonredundant proton–proton contacts in proteins is homonuclear,
doubly heteronuclear-edited <sup>1</sup>H–<sup>1</sup>H 4D
spectra.<sup><xref ref-type="bibr" rid="ref53">53</xref>,<xref ref-type="bibr" rid="ref58">58</xref>,<xref ref-type="bibr" rid="ref59">59</xref></sup> Severe signal
truncation in the presence of three indirect dimensions can be circumvented
by nonuniform sampling (NUS) of only a subset of the indirect points
by covering a sufficiently large Nyquist grid, which is needed to
obtain narrow line widths.<sup><xref ref-type="bibr" rid="ref60">60</xref>,<xref ref-type="bibr" rid="ref61">61</xref></sup> Given that in the solid
state, spectral acquisition is usually dominated by sensitivity rather
than resolution, NUS with high sparsity has rarely been implemented.
However, increased sensitivity is obtained through predominant sampling
of higher signal-to-noise data found at short indirect evolution times
as well.<sup><xref ref-type="bibr" rid="ref62">62</xref></sup> This is pronounced for 4D approaches,
where the enhancement is obtained in three indirect dimensions.<sup><xref ref-type="bibr" rid="ref61">61</xref></sup> Poisson distribution of sampling gaps<sup><xref ref-type="bibr" rid="ref63">63</xref></sup> and reconstruction by the iterative soft thresholding
(hmsIST) algorithm<sup><xref ref-type="bibr" rid="ref64">64</xref></sup> turns out to be a
powerful means for achieving both improvements in resolution and signal-to-noise
in solid-state NMR spectroscopy.</p><p>Accurate and unambiguous structural
restraints are demonstrated
here from 4D NUS spectra with ultrasparse Poisson-gap sampling and
diagonal compensation. We show that the near absence of diagonal peaks
enables artifact-free spectra despite nonuniform sampling at an ultrasparse
sampling rate of as low as 2%. The diagonal is suppressed by subtraction
of compensating scans recorded without mixing in only a fraction of
the time used for the regular spectrum, resulting in identification
of cross-peaks that would otherwise be obscured by the diagonal.</p></sec><sec id="sec2"><title>Experimental Section</title><sec id="sec2.1"><title>Sample Preparation</title><p>The two proteins used for this study
are the SH3 domain of α-spectrin and EAS<sub>Δ15</sub> hydrophobin rodlets. Samples were prepared as described previously
using 25% and 100% <sup>1</sup>H back-substitution of amide protons,
respectively, in otherwise deuterated, <sup>13</sup>C and <sup>15</sup>N-labeled protein.<sup><xref ref-type="bibr" rid="ref32">32</xref>,<xref ref-type="bibr" rid="ref65">65</xref></sup> Hence, methyl groups only bear
protons to the extent defined by incompleteness of commercially available
deuterated D<sub>2</sub>O and glucose (97% <sup>2</sup>D) here, corresponding
to an abundance of ∼9%. In the case of the SH3 domain, paramagnetic
doping was applied as described previously using 100 mM [Cu<sup>II</sup>(edta)]<sup>2–</sup>.<sup><xref ref-type="bibr" rid="ref66">66</xref></sup> In the
case of the hydrophobin, rodlets were formed in the absence of doping
agent and subsequently incubated overnight in a 75 mM [Cu<sup>II</sup>(edta)]<sup>2–</sup> solution. For the SH3 domain, approximately
3.5 mg was centered in a 2.5 mm rotor using self-made spacers of Teflon
tape in the top and bottom of the rotor. For the hydrophobin rodlets,
roughly 1 mg protein was spun into a 1.3 mm rotor and sealed with
fluorinated rubber plugs to prevent loss of water.</p></sec><sec id="sec2.2"><title>Spectral Acquisition</title><p>SH3 spectra were recorded at 700
MHz Larmor frequency using a Bruker Avance narrow-bore spectrometer
with a triple-resonance probehead. Magic Angle Spinning was set to
25-kHz rotation at approximately 10 °C. Interscan delays were
chosen to be 0.5 s.</p><p>Spectral widths in indirect dimensions were
set to 12 and 26 ppm for <sup>1</sup>H and <sup>15</sup>N/<sup>13</sup>C with indirect evolution times of 5 ms in each case. Mixing was
achieved using 8 ms RFDR<sup><xref ref-type="bibr" rid="ref67">67</xref></sup> with 180°
pulses of 4.7 μs (ν<sub>RF</sub> = 106 kHz). In a time-shared<sup><xref ref-type="bibr" rid="ref68">68</xref></sup> 4D experiment, methyl resonances in indirect
heteronuclear dimensions can be separated from the amide signals by
their corresponding proton chemical shift if the indirect <sup>1</sup>H spectral width is large enough to avoid folding (here 12 ppm).
Hydrophobin spectra were recorded as nontime-shared versions in a
1.3 mm rotor at 1 GHz Larmor frequency and 60 kHz MAS at approximately
20 °C. Indirect spectral widths were set to 4.6 and 28.6 ppm
for <sup>1</sup>H and <sup>15</sup>N, respectively. Carriers were
set to the center of the amide bulk in both cases. Mixing was achieved
using 6.7 ms RFDR with 180° pulses of 2.5 μs (ν<sub>RF</sub> = 200 kHz). Four identical data sets with RFDR mixing were
recorded, and one data set was recorded in the absence of mixing.
Each data set was shifted independently to compensate for drift of
the <italic>B</italic><sub>0</sub> field before further processing.</p><p>Figure <xref rid="fig1" ref-type="fig">1</xref> depicts a 4D experimental scheme
using dipolar transfers between protons and heteronuclei and time-shared<sup><xref ref-type="bibr" rid="ref68">68</xref></sup> indirect heteronuclear evolution periods. The
pulse program used (for Bruker software) can be found on the Linser
group webpage (<uri xlink:href="http://mpibpc.mpg.de/linser">mpibpc.mpg.de/linser</uri>) or can be obtained
from the authors. Incrementation of <sup>13</sup>C is scaled down
by a factor of 2.5 by temporary storage of <sup>13</sup>C magnetization
along the axis of <italic>B</italic><sub>0</sub> after 40% of the
incremented time. This leads to correct scaling (in ppm) when treating
the indirect dimensions as <sup>15</sup>N and also serves to keep
the evolution time short with respect to evolution of <sup>13</sup>C–<sup>13</sup>C scalar couplings. Nonuniform sampling for
4D experiments was implemented via a variable counter (vc) list with
a predefined 2% schedule. To avoid aliasing artifacts, sampling gaps
should be kept small but as random as possible, which is achieved
by Poisson distribution centered at zero value.<sup><xref ref-type="bibr" rid="ref63">63</xref></sup> We recorded eight successive scans according to States-TPPI
phase-sensitive incrementation of the three indirect dimensions for
each set of indirect evolution increments.</p><p>The 2D reference
spectrum plotted under the 4D planes in Figure <xref rid="fig2" ref-type="fig">2</xref>A,B and <xref rid="notes-2" ref-type="notes">Supporting Information,
Figures 1 and 6</xref> was recorded using the same (truncated) scheme
without F1 incrementation and without mixing.</p><p>2D spectra were
processed using Topspin, and 4D data was processed
using NMRpipe.<sup><xref ref-type="bibr" rid="ref69">69</xref></sup> For the 4D spectra, Fourier
transformation of the direct dimension was followed by the hmsIST
routine<sup><xref ref-type="bibr" rid="ref64">64</xref></sup> with 200 iterations and subsequent
reshuffling of transients for phase-sensitive recording and time increments
in order to allow for standard FFT of the indirect dimensions. Apodization
was performed using a sine bell shifted by π/2 for all dimensions
and 2048 × 64 × 64 × 64 points for the direct and indirect
ones. The nmrPipe processing scripts are provided on the Linser group
webpage. The hmsIST routine<sup><xref ref-type="bibr" rid="ref64">64</xref></sup> as well as
a sampling schedule generator can be obtained on the Wagner lab webpage
(<uri xlink:href="http://gwagner.med.harvard.edu/intranet/hmsIST/">gwagner.med.harvard.edu/intranet/hmsIST/</uri>).</p><p>Spectra
were converted to ucsf format with the routine included
in the Sparky program<sup><xref ref-type="bibr" rid="ref70">70</xref></sup> and used for analysis
in CcpNmr.<sup><xref ref-type="bibr" rid="ref71">71</xref></sup></p><p>Assigned 4D HN-HN cross-peaks
were converted into distance restraints
with a uniform calibration procedure to convert peak intensity into
distance. Structure ensembles were calculated with a simulated annealing
procedure implemented in ARIA<sup><xref ref-type="bibr" rid="ref72">72</xref></sup>/CNS<sup><xref ref-type="bibr" rid="ref73">73</xref></sup>. Backbone dihedral angle restraints were predicted
from chemical shifts<sup><xref ref-type="bibr" rid="ref74">74</xref></sup> and used in addition
of the HN-HN distance restraints. Structure calculations and distances
calibration are presented in detail in the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>.</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Practical implementation of diagonal-free 4D proton–proton
correlations. Pulse scheme for NUS time-shared HXXH 4D correlations.
Large brackets indicate the HXH pathway extractable for 3D HXH correlations.
Mixing via RFDR is elicited by <italic>n</italic><sub>i</sub> = {<italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, <italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, 0} and <italic>n</italic><sub>i</sub> = {<italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, <italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, <italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, <italic>t</italic><sub>mix</sub>/τ<sub>rot</sub>, 0} for successive scans in
the case of 25% and 100% <sup>1</sup>H back-exchange, respectively.
Time-shared evolution (by including the small brackets) is provided
by simultaneous H/X cross-polarization transfer steps for amides and
methyl groups and storage of <sup>13</sup>C magnetization to achieve
scaling of indirect evolution increments.<sup><xref ref-type="bibr" rid="ref44">44</xref>,<xref ref-type="bibr" rid="ref68">68</xref></sup> Phases of the first proton πι/2 pulse and the CP pulses
marked by asterisks were cycled for phase-sensitive incrementation.</p></caption><graphic xlink:href="ja-2014-04603g_0001" id="gr1" position="float"></graphic></fig></sec></sec><sec sec-type="results" id="sec3"><title>Results</title><p>In contrast
to solution NMR approaches,<sup><xref ref-type="bibr" rid="ref49">49</xref>−<xref ref-type="bibr" rid="ref53">53</xref></sup> we find that diagonal compensation in the solid state
can be achieved with little sensitivity loss compared with the noncompensated
spectrum. For SH3 and hydrophobin, only ∼27% and ∼13%
of the sensitivity needs to be sacrificed for diagonal compensation.
The reason is that in the absence of dipolar mixing (i.e., in scans
that can be subtracted for elimination of the diagonal), signal intensity
is significantly higher (see below) relative to the regular scans
that include the desired information (regular scans refer to scans
carrying both diagonal and cross peaks). This effect is due to pulse
imperfections, relaxation, and rf-inhomogeneity during the dipolar
mixing sequence in the latter. In addition, the diagonal signals become
significantly dispersed into cross-peak signals in the course of magnetization
transfer to nearby nuclei. Accordingly, highest sensitivity is obtained
by recording and subtracting single diagonal-only compensating scans
from the sum of multiple regular transients. For the SH3 domain with
25% proton back-exchange, diagonal signal in compensation scans is
approximately three times more intense than in the regular scans on
average. This allows experiments of highest signal-to-noise with an
accordingly low number of interleaved compensation transients. When
optimized, 27% of the sensitivity is sacrificed (see <xref rid="notes-2" ref-type="notes">Supporting Information</xref> for more details). Hydrophobin spectra
were recorded with a 100% proton back-protonation at 60 kHz MAS using
a 1 GHz spectrometer. For this labeling scheme, diagonal-signal intensity
is expectedly lower than obtained with stochastic partial protonation.
Hardly any price has to be paid here in terms of additional measurement
time since a very low number of compensation scans is necessary to
eliminate the smaller diagonal in this case. Diagonal signal in compensation
scans is here more than six times more intense than in the regular
scans on average. One scan without mixing was recorded here for every
4 regular scans with recoupling and required further downscaling to
0.6 upon processing. This is commensurate with an additional time
requirement of 14% for diagonal compensation. Due to site-specific
differences in protein mobility and the density of the proton dipolar-coupling
network, small residual diagonal peaks are observed for a subset of
the protein residues after compensation with intensities on the order
of weak cross peaks as expected.</p><p>Given the much smaller sensitivity
loss for tailored compensation
approaches in the solid state compared to solution NMR, zero-quantum
(ZQ) recoupling techniques can be implemented without diagonal-related
problems at low cost. ZQ recoupling sequences like RFDR<sup><xref ref-type="bibr" rid="ref67">67</xref></sup> have been used much for proton–proton
recoupling in deuterated solids.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref34">34</xref>,<xref ref-type="bibr" rid="ref44">44</xref>,<xref ref-type="bibr" rid="ref75">75</xref></sup> This common use for
informative distance restraints in structure calculations is further
facilitated when diagonal signal is suppressed.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>4D spectroscopy and diagonal
compensation. (A) A representative
F3(<sup>15</sup>N)/F4(<sup>1</sup>H) 2D slice as an excerpt of a NUS
diagonal-free 4D experiment (black contours), with indirect F1(<sup>1</sup>H)/F2(<sup>15</sup>N) chemical shifts set as indicated by
dashed lines, showing spatial contacts of K26 H<sup>N</sup>. *L61
is a bleed-through and has maximum intensity at different F1/F2 shifts.
(B) The nondiagonal-free spectrum at identical shifts. In both slices
the reference 2D HN correlation (cyan contours) is overlaid for better
overview. (C) Neighboring amides of K26 as a representative residue
as seen in the crystal structure of the SH3 domain of α-spectrin
(PDB: 2NUZ)
and their distances to K26 H<sup>N</sup>. D) Traces along <italic>F3</italic> through K26/K27 and K26/A12 cross peaks (as shown by
arrows in A and B), which are in relative proximity to the diagonal.
Whereas K27 peak intensity (traces b vs d) is doubled in comparison
to its expected value, intensities of distant A12 (traces c vs a)
are fully dominated by the diagonal. The same is true also for A11
(as can be seen in E). (E) 4D peak volumes extracted from the respective
2D slice and plotted over their corresponding internuclear distances
(extracted from X-ray structure 2NUZ). Peaks significantly deviating from
a consistent distance/intensity relation (delineated by the dashed
line) incur erroneous distance restraints. The error introduced by
noise is on the order of the symbol sizes used. (F) The set of resonances
recorded in regular homonuclear correlation experiments consist of
amide/amide cross peaks, whereas only one peak per amino acid pair
is observed. Thus, every cross peak contains useful and nonredundant
information. For time-shared versions and adequate protein labeling,
additional correlations are recorded involving methyls. This 2D plane
was recorded using the shortened version of the pulse scheme shown
in Figure <xref rid="fig1" ref-type="fig">1</xref>. Negative contours are shown in
red. See <xref rid="notes-2" ref-type="notes">Supporting Information, Figure 2</xref> for the same spectrum without diagonal compensation.</p></caption><graphic xlink:href="ja-2014-04603g_0002" id="gr2" position="float"></graphic></fig><p>We generated diagonal-compensated NUS HXXH 4D experiments
according
to the scheme in Figure <xref rid="fig1" ref-type="fig">1</xref>. Both the moiety
from which magnetization is transferred and the moiety where it can
be detected after mixing are encoded using a proton and a heteronuclear
chemical shift evolution. For a structure of the well-characterized
SH3 domain of α-spectrin based on sparse amide restraints, we
employed ultrasparse (2%) Poisson-gap sampling, diagonal compensation
(one out of three transients), and two heteronuclear time-shared dimensions.
In this way spectra with excellent resolution were obtained within
4 days of total measurement time. Ultrasparse sampling allows flexible
choices for numbers of regular and compensation scans even in the
4D. The spectra obtained (Figure <xref rid="fig2" ref-type="fig">2</xref>A,F), provide
high resolution as well as signal-to-noise ratios without potential
artifacts. Also, due to the absence of diagonal signals and the performance
of the hmsIST reconstruction, there is no detectable <italic>t</italic><sub>1</sub> noise. Figure <xref rid="fig4" ref-type="fig">4</xref>B–D classify
the kind of restraints obtained. A structure calculation on the basis
of these restraints is described below.</p><p>In the absence of diagonal
compensation, a large region of each
2D slice (on the order of 1.5 and 10 ppm wide for <sup>1</sup>H and <sup>15</sup>N, respectively) would be affected by the presence of the
diagonal in a four-dimensional (4D) experiment. The improvement with
diagonal suppression is shown in Figure <xref rid="fig2" ref-type="fig">2</xref> for
SH3, by comparing the compensated spectrum (Figure <xref rid="fig2" ref-type="fig">2</xref>A) with the regular 4D (Figure <xref rid="fig2" ref-type="fig">2</xref>B).
Without compensation, significant errors in intensity are introduced
for those peaks that are close to the diagonal (Figure <xref rid="fig2" ref-type="fig">2</xref>D). With diagonal compensation, on the other hand, peak intensities
within the set of restraints obtained for a specific residue follow
a consistent trend dependent on the internuclear distance in the majority
of cases (see Figure <xref rid="fig2" ref-type="fig">2</xref>E).</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Diagonal-compensated
4D <sup>1</sup>H/<sup>1</sup>H correlations
in spectra of hydrophobin rodlets lacking crystalline order. (A) Negative-stain
EM image of EAS<sub>Δ15</sub> hydrophobin rodlets. (B) Surface
charge representation of the amphipathic hydrophobin monomer (PDB 2K6A). (C) Hypothetical
model of the rodlet structure as a functional fungal amyloid obtained
using molecular docking with the solution structure of monomeric EAS<sub>Δ15</sub> and mutagenesis and other biophysical data.<sup><xref ref-type="bibr" rid="ref76">76</xref></sup> (D) <sup>1</sup>H amide resolution of the rodlets
(black, bottom) compared to the microcrystalline SH3 domain (gray,
top). Both spectra are shown without apodization or truncation of
the FID. (E and F) Representative 2D slices from an HNNH 4D experiment.
Diagonal compensation was achieved (black contours) applying 1 out
of 5 scans without dipolar recoupling. (Gray contours display noncompensated
scans only, blue contours represent an overlaid reference HN correlation.)
Proximities seen in (E) are expected on the basis of the monomer structure.<sup><xref ref-type="bibr" rid="ref77">77</xref></sup> The peaks labeled in (F) cannot be explained
by the monomer fold and hint to major structural rearrangements upon
rodlet assembly. Hydrophobin spectra were recorded on deuterated hydrophobin
EAS<sub>Δ15</sub> rodlets, 100% <sup>1</sup>H back-exchanged
at labile sites in a 1.3 mm rotor at 1 GHz <sup>1</sup>H Larmor frequency
and 60 kHz MAS. Proton-detected experiments used for obtaining backbone
assignments are described in the Supplement.</p></caption><graphic xlink:href="ja-2014-04603g_0003" id="gr3" position="float"></graphic></fig><p>A benefit of diagonal-compensated HXXH spectroscopy is obtained
for proteins with heterogeneously or homogeneously broadened resonances,
which are particularly affected by diagonal obstructions covering
large parts of the spectral space. <sup>1</sup>H/<sup>1</sup>H proximities
can be derived from application of diagonal-compensated NUS-4D <sup>1</sup>H/<sup>1</sup>H correlations for structural assessment of
hydrophobin rodlets (shown in Figure <xref rid="fig3" ref-type="fig">3</xref>). These
rodlets are partially disordered functional amyloids from fungal spores.<sup><xref ref-type="bibr" rid="ref77">77</xref>,<xref ref-type="bibr" rid="ref78">78</xref></sup> Even though EM pictures (see the negative-stain representation in
Figure <xref rid="fig3" ref-type="fig">3</xref>A) of rodlets formed from the amphipathic
monomer (Figure <xref rid="fig3" ref-type="fig">3</xref>B) are macroscopically well
ordered, the atomic order seems to be limited to a relatively small
portion (<50%) of the protein sequence with a β-sheet character.<sup><xref ref-type="bibr" rid="ref65">65</xref>,<xref ref-type="bibr" rid="ref76">76</xref></sup> (See Figure <xref rid="fig3" ref-type="fig">3</xref>D for a comparison of the <sup>1</sup>H<sup>N</sup> bulk resolution with the microcrystalline SH3
domain.) <sup>13</sup>C resonances from only ∼20 residues in
the well-structured region of the sample could be identified and assigned
to amino acid types using <sup>13</sup>C–<sup>13</sup>C homonuclear
correlation spectroscopy (DARR<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> mixing)
and a 3D NCACX spectrum initially.<sup><xref ref-type="bibr" rid="ref65">65</xref></sup> Here,
the initial noncompensated 4D and 3D <sup>1</sup>H–<sup>1</sup>H spectra were almost entirely noninterpretable, forming the motivation
to seek for general high-signal-to-noise diagonal-free strategies.
Figure <xref rid="fig3" ref-type="fig">3</xref>E,F shows a comparison between standard
and diagonal-free homonuclear correlations in the case of 4D HNNH
correlations. Backbone assignments and structure calculation for this
protein are in progress.</p><p>For the SH3 domain, we generated and
analyzed structures calculated
from sparse 4D restraints (see the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref> for a detailed description). The calculation focuses
on the set of peaks representing correctly assigned amide-to-amide
distances only. (Methyl-amide contacts obtained with this labeling
scheme are much sparser, they are not affected by diagonal signal,
and it has been shown previously that methyl–amide restraints
improve structural quality, see details in the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>. For assignment fidelity effects, see
below.) Here from 161 resolved 4D amide–amide peaks, 99 unique <sup>1</sup>H<sup>N</sup>–<sup>1</sup>H<sup>N</sup> distance restraints
could be extracted (see Figure <xref rid="fig4" ref-type="fig">4</xref>B,C). The final set of nonredundant restraints consists
of 35 sequential (|<italic>i</italic> – <italic>j</italic>|
= 1), 14 short-range (|<italic>i</italic> – <italic>j</italic>| = 2), 5 medium-range (2 < |<italic>i</italic> – <italic>j</italic>| <5), and 45 long-range (|<italic>i</italic> – <italic>j</italic>| > 4) restraints. Similar to methyl NOEs in structure
calculation of large proteins in solution, amide–amide distances
as shown here thus represent structural restraints of particular,
nontrivial character, especially of value for defining β-sheet
and α-helical secondary structures, but also for the overall
protein fold. Figure <xref rid="fig5" ref-type="fig">5</xref>A shows a backbone representation
of the 10 lowest-energy structures based on restraints only from confirmed,
correct amide–amide cross peaks as seen in the diagonal-free
spectrum.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Correlations obtained in homonuclear <sup>1</sup>H–<sup>1</sup>H experiments on perdeuterated SH3. (A) All peak-like intensity
above the noise level, picked at root resonance combinations (H/N
HSQC shift pairs) only and representing potential (but not necessarily
real) cross peaks. Orange shading represents the minimum space affected
by diagonal signal, within which a great part of all peak-like intensity
actually represents artifact peaks. Note the logarithmic scale of
the <italic>Y</italic>-axis. Error bars refer to the noise level in
the spectrum. Peak intensities were normalized using the F52/K26 cross
peak as a representative peak, well separated from the diagonal. (B)
Numbers of amide–amide contacts observed for a 4D version recorded
on the SH3 domain of α-spectrin (black bars). Less peak intensity
and fewer contacts are seen in the flexible loop regions (orange shades).
Most contacts are long-range contacts and thus of high benefit for
structure calculation. One asterisk indicates Pro residues, two asterisks
denote those residues which are exchange-broadened in HN correlations
and/or have not be assigned unambiguously. (C) Contact map of all
visible correlations (cyan rectangles) overlaid on all residue pairs
within 7.5 Å (gray rectangles). (D) Classification of the extracted
(nonredundant) distance restraints.</p></caption><graphic xlink:href="ja-2014-04603g_0004" id="gr4" position="float"></graphic></fig><p>At the level of data quality obtained, the structural quality
(with
a backbone precision of 1.28 ± 0.21 Å) is very close to
the potential optimum expected for the kind and number of restraints
used: Using the same set of amide–amide restraints, but with
“true” crystal-structure distances, a backbone precision
of 1.15 ± 0.22 Å would be obtained.</p><p>The improvement
in the backbone fold accuracy (from 2.05 to 1.69
Å) upon structure calculation with diagonal compensation in comparison
to a calculation without compensation seems modest (RMSDs with respect
to the crystal-structure, see also <xref rid="notes-2" ref-type="notes">Supporting
Information, Figure 10</xref>). The structural restraints that this
comparison is based on, however, differ here only due to improved
integration of cross-peaks, whereas the kinds of restraints taken
into account are identical and only from correctly assigned peaks
in both calculations. The quality of <italic>de novo</italic> structures
obtained from noncompensated spectra only, on the other hand, would
be dominated by the lower reliability of identification and assignment
of cross peaks. The probability for correct peak selection is difficult
to quantify. Nevertheless, significant qualitative differences in
spectral interpretability are evident from the relative space covered
by diagonal signal.</p><p>In addition, Figure <xref rid="fig4" ref-type="fig">4</xref>A shows peak-like intensity
in a 4D experiment plotted over the distance of each of those signals
to the diagonal. Peak intensity is taken to be the intensity above
the noise level at all H/N/N/H combinations at which cross peaks could
potentially be located on the basis of HSQC root resonances. Sufficiently
distant from the diagonal, these data purely represent real cross
peaks of structural relevance. With less separation of cross-peaks
from the diagonal, however, unreal “peaks” show up that
would not be expected on the basis of atomic distances. These false
peaks are inadvertently introduced into structure calculations for
proteins of unknown structure (particularly for automated peak picking)
unless large areas around the diagonal are avoided for peak picking
or circumvented by diagonal compensation.</p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Structure calculation
from <sup>1</sup>H–<sup>1</sup>H restraints.
(A) Backbone representation of the structural ensembles obtained from
diagonal-free 4D correlations superimposed on the reference X-ray
structure (PDB: 2NUZ, shown in red). Top: Calculation using peak integrals from diagonal-compensated
spectra; bottom: using integrals of the same peaks without compensation.
(B) Representation of backbone structure accuracy (bias to the X-ray
structure 2NUZ, red) and precision (blue, RMSD to mean) based on high-quality diagonal-free
4D restraints (solid lines). Dashed lines correspond to structures
from noncompensated restraints. Secondary structure elements are shown
on top with red arrows (strands) and a blue bar (helix). The flexible
termini and loop regions are indicated by brown shades, where few
contacts are available. All other regions are not far from 1 Å
resolution. (C) Comparison of backbone structure accuracy (bias to
the X-ray structure 2NUZ) using additional methyl restraints (blue) and using amide–amide
contacts only (red). All-atom RMSDs (black) are similar to backbone
RMSDs in the presence of amide–methyl restraints (bullets),
demonstrating the general potential of methyl-based restraints to
further improve accuracy. Methyl data shown here for comparison were
acquired using the time-shared experiment in its (truncated) 3D version.
See the <xref rid="notes-2" ref-type="notes">Supporting Information</xref> for details
on methyl data.</p></caption><graphic xlink:href="ja-2014-04603g_0005" id="gr5" position="float"></graphic></fig></sec><sec sec-type="discussion" id="sec4"><title>Discussion</title><p>Distances
based on unambiguous amide–amide spectra are valuable
for identification of a backbone fold and can be supplemented by methyl–methyl<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> and methyl–amide<sup><xref ref-type="bibr" rid="ref44">44</xref></sup> restraints to provide high-resolution structures if these are suitably
labeled. A more comprehensive set of distances may be obtained by
incorporation of other side chain protons in partially or fully side
chain-protonated samples,<sup><xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref30">30</xref>,<xref ref-type="bibr" rid="ref36">36</xref>,<xref ref-type="bibr" rid="ref79">79</xref></sup> however, increasing numbers of
peaks will always also lead to increasing ambiguity in assignment
and structural data. Different kinds of structural data obtained with
different approaches are usually not mutually exclusive and can be
combined with each other.<sup><xref ref-type="bibr" rid="ref23">23</xref>,<xref ref-type="bibr" rid="ref24">24</xref>,<xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref80">80</xref>,<xref ref-type="bibr" rid="ref81">81</xref></sup> If resolved <sup>13</sup>C-edited distance restraints from protonated
samples are available, which is less likely for increasing molecular
weight, these can always be integrated into the structure calculation
as unambiguous or ambiguous<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> restraints.
Where better suited, paramagnetic restraints<sup><xref ref-type="bibr" rid="ref82">82</xref></sup> and surface contacts<sup><xref ref-type="bibr" rid="ref83">83</xref></sup> can be easily
combined with proton-detected methods.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup></p><p>Ultrasparse NUS 4D HXXH spectra turn out to be highly reliable
in terms of unambiguous peak identification and assignment and provide
sparse and nonredundant long-range distance restraints. This makes
them a well suited basis for proton-distance-based structure assessment
of proteins. In contrast to solution NMR, even though generally much
sharper peaks are obtained and significant sacrifice of sensitivity
results from both subtraction of transients<sup><xref ref-type="bibr" rid="ref49">49</xref></sup> as well as from elimination of coherences,<sup><xref ref-type="bibr" rid="ref53">53</xref></sup> diagonal-compensated 4D spectroscopy in the solid state has not
been used so far for structure elucidation. Providing a valuable tool
with respect to the increasing use of proton spins in the solid state,
this or modified approaches have the potential to facilitate acquisition
of structure-relevant data on deuterated samples with different degrees
of protons selectively introduced at various protein sites.</p><p>Whereas amide and methyl restraints are able to mostly deliver
a reliable protein fold, dilute side-chain protonation in terms of
random adjoining protonation (RAP labeling)<sup><xref ref-type="bibr" rid="ref28">28</xref></sup> is expected to be of major advantage for correct side chain orientation
and packing in future studies. For this and stochastic labeling generally
(implying a reduced labeling percent causing larger diagonal to cross-peak
ratios), diagonal compensation will be very well suited. Also for
heterogeneous preparations, as shown for (but not limited to) hydrophobins
with a 100% back-protonation at 60 kHz MAS and 1 GHz Larmor frequency,
significant benefits of diagonal compensation become obvious from
the average <sup>1</sup>H line width of ca. 0.25 ppm (250 Hz), which
translates into short indirect acquisition times and a significant
portion of the total spectral space to practically be covered by diagonal
signal. Thus, spectral interpretability as well as distance-restraint
data quality is significantly increased upon diagonal compensation.</p><p>In agreement to previous studies,<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref34">34</xref></sup> the efficiency
of RFDR recoupling was not seen to be compromised by fast spinning
and accordingly short pulse widths (2.5 μs 180°-pulse length
at a rotor period of 16.7 μs). We used RFDR as a robust and
easy-to-implement scheme with a high relative signal-to-noise even
after diagonal suppression. It is also compatible with the large <sup>1</sup>H bandwidths required for time-shared (amide–methyl)
spectra. In principle, however, RFDR could be replaced by other robust
and well-working zero- or double-quantum recoupling schemes when requirements
for <sup>1</sup>H rf power and bandwidths are met. For spectra with
small diagonal-to-cross peak intensity ratios, negligible costs of
measurement time apply for acquisition of compensation scans, so acquisition
of compensation scans can always be easily implemented. Better data
quality and interpretability with compensation can then save a multitude
of the additional measurement time at the analysis and calculation
state.</p><p>Spin-diffusion processes and dipolar truncation effects
are expected
to be more significant for increasing back-substitution levels at
amide sites.<sup><xref ref-type="bibr" rid="ref57">57</xref>,<xref ref-type="bibr" rid="ref84">84</xref></sup> These issues lead, respectively,
to less reliable and fewer long-range restraints for fully back-exchanged
or stoichiometrically methyl-labeled samples. On the other hand, nanomolar
sample amounts of stoichiometrically amide-protonated proteins can
be employed here due to greater overall sensitivity when 1.3 mm rotors
and 100% proton back-substitution at 60 kHz are used. This has advantages
when samples are particularly difficult to make or costly (like for
CHD<sub>2</sub>-methyl labeling).</p><p>Both for 25% <sup>1</sup>H
back-exchange and intermediate MAS as
well as 100% back-exchange and ultrafast spinning, correlations of
the kind shown are straightforward and will be useful in future studies.
This is true even despite the lower number of expected contacts for
100% back-exchange at 60 kHz.</p></sec><sec sec-type="conclusions" id="sec5"><title>Conclusions</title><p>We have shown that the
approach presented here yields proton–proton
distance restraints for partially deuterated proteins and represents
a reduced set of structural data that are sparse in quantity but highly
nonredundant and accurate. Importantly, structural restraints can
be correctly identified and evaluated. We obtain highly resolved correlations
in conjunction with very good sensitivity from diagonal-compensated, <sup>1</sup>H, <sup>15</sup>N/<sup>13</sup>C, <sup>15</sup>N/<sup>13</sup>C, <sup>1</sup>H-edited 4D experiments with ultrasparse Poisson-gap
sampling. Fully demonstrated here using the small protein SH3 and
a relatively well-dispersed spectrum, it also becomes evident that
the reduction in signal overlap is a critical advancement in facilitating
the characterization of challenging targets like the more heterogeneous
hydrophobin rodlets. An SH3 structure based on restraints obtained
using this approach is characterized by a relatively high accuracy
with respect to the minimal set of unambiguous structural restraints
with mainly long-range character. This opens the door for successful
structure assessment of large or poorly ordered proteins.</p><p>We
believe that the quality of data obtainable, in combination
with the simplicity and efficacy of the concept, will lead to proton-based
structure elucidation as a major anchor point in the evolving field
of fast-MAS solid-state NMR. This applies in particular to the steadily
increasing MAS frequencies under which detection of side chain protons
can be resolved without extensive deuteration. The methodologies are
expected to be useful for improved structural characterization of
membrane proteins and fibrillar or prion proteins.</p></sec></body><back><notes id="notes-2" notes-type="si"><title>Supporting Information Available</title><p>Time-shared spectral
data,
effects of diagonal compensation for spinning side bands, sensitivity
considerations, bidirectionality in 4D spectra, structure calculation,
and distance-integral correlations. This material is available free
of charge via the Internet at <uri xlink:href="http://pubs.acs.org">http://pubs.acs.org</uri>.</p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="ja504603g_si_001.pdf"><caption><p>ja504603g_si_001.pdf</p></caption></media></supplementary-material></sec><notes id="notes-1"><title>Author Present Address</title><p><sup>#</sup> Technische Universität München, Germany,
85748 München,
Germany.</p></notes><notes id="NOTES-d1582e1189-autogenerated" notes-type="conflict-of-interest"><p>The
authors declare no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>We thank Drs. J. Hook, D. Thomas, D. Lawes,
and A. Rawal from
the NMR facility at University of New South Wales for spectrometer
access and assistance and Dr. Jim Sun, Harvard Medical School, for
initial help with NMRpipe. We are particularly grateful for Prof.
Dr. Bernd Reif, TU München, for generously providing SH3 protein
for study in Australia and to Dr. Anthony Duff and Karyn Wilde for
assistance with hydrophobin production. The project was funded by
the Australian Research Council (LP0776672 and DP0879121) and ANSTO
Bragg Institute (NDF 1668), the Agilent Thought Leader Award and the
NIH grant GM047467 (to G.W.). We are grateful for support from TGIR-RMN-THC
Fr3050 CNRS. M.S. was supported by a National Health and Medical Research
Council RD Wright Career Development Fellowship, V.M. was supported
by a University of Sydney Vice-Chancellor’s Research Scholarship,
L.B.A. was supported by a EU Marie-Curie IIF Fellowship, and R.L.
acknowledges an Australian Research Council Discovery Early Career
Research Award and a Liebig Fellowship from the Verband der Chemischen
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