Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Identifieur interne : 000254 ( Pmc/Corpus ); précédent : 000253; suivant : 000255Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Auteurs : Ling-Hon Matthew Chu ; Wai-Yan Choy ; Sau-Na Tsai ; Zihe Rao ; Sai-Ming NgaiSource :
- Protein Science : A Publication of the Protein Society [ 0961-8368 ] ; 2006.
Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CLpro. Six target amino acid positions flanking the SARS-CoV 3CLpro cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CLpro. Susceptibility of each peptide substrate to SARS-CoV 3CLpro cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CLpro-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CLpro that is facing the P1′ position is suggested to be occupied by acidic amino acids, thus the P1′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or β-branching amino acids in P3 and P2′ positions may also hinder the binding of SARS-CoV 3CLpro. This study generates a comprehensive overview of SARS-CoV 3CLpro substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CLpro inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.
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DOI: 10.1110/ps.052007306
PubMed: 16600962
PubMed Central: 2242481
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry</title><author><name sortKey="Chu, Ling Hon Matthew" sort="Chu, Ling Hon Matthew" uniqKey="Chu L" first="Ling-Hon Matthew" last="Chu">Ling-Hon Matthew Chu</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation></author><author><name sortKey="Choy, Wai Yan" sort="Choy, Wai Yan" uniqKey="Choy W" first="Wai-Yan" last="Choy">Wai-Yan Choy</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation></author><author><name sortKey="Tsai, Sau Na" sort="Tsai, Sau Na" uniqKey="Tsai S" first="Sau-Na" last="Tsai">Sau-Na Tsai</name><affiliation><nlm:aff id="aff2">Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong</nlm:aff></affiliation></author><author><name sortKey="Rao, Zihe" sort="Rao, Zihe" uniqKey="Rao Z" first="Zihe" last="Rao">Zihe Rao</name><affiliation><nlm:aff id="aff3">Laboratory of Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, 100084 Beijing, China</nlm:aff></affiliation></author><author><name sortKey="Ngai, Sai Ming" sort="Ngai, Sai Ming" uniqKey="Ngai S" first="Sai-Ming" last="Ngai">Sai-Ming Ngai</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">16600962</idno><idno type="pmc">2242481</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242481</idno><idno type="RBID">PMC:2242481</idno><idno type="doi">10.1110/ps.052007306</idno><date when="2006">2006</date><idno type="wicri:Area/Pmc/Corpus">000254</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000254</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry</title><author><name sortKey="Chu, Ling Hon Matthew" sort="Chu, Ling Hon Matthew" uniqKey="Chu L" first="Ling-Hon Matthew" last="Chu">Ling-Hon Matthew Chu</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation></author><author><name sortKey="Choy, Wai Yan" sort="Choy, Wai Yan" uniqKey="Choy W" first="Wai-Yan" last="Choy">Wai-Yan Choy</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation></author><author><name sortKey="Tsai, Sau Na" sort="Tsai, Sau Na" uniqKey="Tsai S" first="Sau-Na" last="Tsai">Sau-Na Tsai</name><affiliation><nlm:aff id="aff2">Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong</nlm:aff></affiliation></author><author><name sortKey="Rao, Zihe" sort="Rao, Zihe" uniqKey="Rao Z" first="Zihe" last="Rao">Zihe Rao</name><affiliation><nlm:aff id="aff3">Laboratory of Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, 100084 Beijing, China</nlm:aff></affiliation></author><author><name sortKey="Ngai, Sai Ming" sort="Ngai, Sai Ming" uniqKey="Ngai S" first="Sai-Ming" last="Ngai">Sai-Ming Ngai</name><affiliation><nlm:aff id="aff1">Molecular Biotechnology Program</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong</nlm:aff></affiliation></author></analytic><series><title level="j">Protein Science : A Publication of the Protein Society</title><idno type="ISSN">0961-8368</idno><idno type="eISSN">1469-896X</idno><imprint><date when="2006">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL<sup>pro</sup>) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL<sup>pro</sup>. Six target amino acid positions flanking the SARS-CoV 3CL<sup>pro</sup> cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL<sup>pro</sup>. Susceptibility of each peptide substrate to SARS-CoV 3CL<sup>pro</sup> cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL<sup>pro</sup>-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CL<sup>pro</sup> that is facing the P1′ position is suggested to be occupied by acidic amino acids, thus the P1′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or β-branching amino acids in P3 and P2′ positions may also hinder the binding of SARS-CoV 3CL<sup>pro</sup>. This study generates a comprehensive overview of SARS-CoV 3CL<sup>pro</sup> substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL<sup>pro</sup> inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Protein Sci</journal-id><journal-title>Protein Science : A Publication of the Protein Society</journal-title><issn pub-type="ppub">0961-8368</issn><issn pub-type="epub">1469-896X</issn><publisher><publisher-name>Cold Spring Harbor Laboratory Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16600962</article-id><article-id pub-id-type="pmc">2242481</article-id><article-id pub-id-type="publisher-id">699</article-id><article-id pub-id-type="doi">10.1110/ps.052007306</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chu</surname><given-names>Ling-Hon Matthew</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="fn1">4</xref></contrib><contrib contrib-type="author"><name><surname>Choy</surname><given-names>Wai-Yan</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="fn1">4</xref></contrib><contrib contrib-type="author"><name><surname>Tsai</surname><given-names>Sau-Na</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Rao</surname><given-names>Zihe</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Ngai</surname><given-names>Sai-Ming</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Molecular Biotechnology Program</aff><aff id="aff2"><label>2</label>Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong</aff><aff id="aff3"><label>3</label>Laboratory of Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, 100084 Beijing, China</aff><author-notes><fn id="fn1"><label>4</label><p>These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="ppub"><month>4</month><year>2006</year></pub-date><volume>15</volume><issue>4</issue><fpage>699</fpage><lpage>709</lpage><history><date date-type="received"><day>6</day><month>12</month><year>2005</year></date><date date-type="rev-recd"><day>6</day><month>12</month><year>2005</year></date><date date-type="accepted"><day>13</day><month>1</month><year>2006</year></date></history><permissions><copyright-statement>Copyright © 2006 The Protein Society</copyright-statement></permissions><abstract><p>Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL<sup>pro</sup>) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL<sup>pro</sup>. Six target amino acid positions flanking the SARS-CoV 3CL<sup>pro</sup> cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL<sup>pro</sup>. Susceptibility of each peptide substrate to SARS-CoV 3CL<sup>pro</sup> cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL<sup>pro</sup>-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CL<sup>pro</sup> that is facing the P1′ position is suggested to be occupied by acidic amino acids, thus the P1′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or β-branching amino acids in P3 and P2′ positions may also hinder the binding of SARS-CoV 3CL<sup>pro</sup>. This study generates a comprehensive overview of SARS-CoV 3CL<sup>pro</sup> substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL<sup>pro</sup> inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.</p></abstract><kwd-group><kwd>substrate specificity</kwd><kwd>SARS-CoV</kwd><kwd>protease</kwd><kwd>MALDI-TOF</kwd><kwd>mass spectrometry</kwd><kwd>synthetic peptide</kwd></kwd-group></article-meta></front><floats-wrap><fig position="float" id="f1"><label>Figure 1</label><caption><p>Primary screening: Mass spectrometry analysis of SARS-CoV 3CL<sup>pro</sup> cleavage products. (<italic>A</italic>) Positive control. PS01 peptide peak (*) was resolved before cleavage assay (<italic>A-1</italic>) and was absent from the mass spectrum after the reaction (<italic>A-2</italic>). (<italic>B</italic>) P2 position. For PS02 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>B-1</italic>), and only the peptide peaks corresponding to Leu (*) and Phe (**) substitutions in the P2 position were absent, whereas all other peaks remained after the cleavage reaction (<italic>B-2</italic>). (<italic>C</italic>) P3 position. For PS03 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>C-1</italic>), and only the peptide peak with Pro (*) substitution in the P3 position remained after the reaction (<italic>C-2</italic>). (<italic>D</italic>) P4 position. For PS04 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>D-1</italic>), which were all absent from the mass spectrum after the reaction (<italic>D-2</italic>). (<italic>E</italic>) P1′ position. For PS05 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>E-1</italic>), and the peptide peaks with Pro (*), Asp (**), and Glu (***) in the P1′ position remained after the reaction (<italic>E-2</italic>). (<italic>F</italic>) P2′ position. For PS06 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>F-1</italic>), and the peptide peaks with Pro (*) and Ile/Leu (**) in the P2′ position remained after the reaction (<italic>F-2</italic>). (<italic>G</italic>) P3′ position. For PS07 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (<italic>G-1</italic>), which were all absent from the mass spectrum after the reaction (<italic>G-2</italic>).</p></caption><graphic xlink:href="699fig1"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><p>Amino acid sequence of the seven synthetic peptide substrates</p></caption><graphic xlink:href="699tbl1"></graphic><table-wrap-foot><fn><p><sup>a</sup> The amino acids flanking the SARS-CoV 3CL<sup>pro</sup> cleavage sites are labeled from the amino terminus to the carboxyl terminus as follows: -P3-P2-P1 ↓ P1′-P2′-P3′, as described previously (Schechter and Berger 1967).</p><p><sup>b</sup> The control peptide substrate is based on the amino acid sequence of one of the SARS-CoV 3CL<sup>pro</sup> cleavage sites on the SARS-CoV BJ01 polyprotein pp1ab (residues 3232–3247).</p><p><sup>c</sup> The corresponding amino acid at this position is substituted by a mixture of 20 standard amino acids in equal molar ratios.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>Table 2</label><caption><p>Summary of results from primary screening</p></caption><graphic xlink:href="699tbl2"></graphic><table-wrap-foot><fn><p><sup>a</sup> The positions flanking the SARS-CoV 3CL<sup>pro</sup> cleavage sites are labeled from the N terminus to the C terminus as follows: -P3-P2-P1 ↓ P1′-P2′-P3′, as described previously (Schechter and Berger 1967).</p><p><sup>b</sup> The original amino acid residue in the position under investigation before the substitution with 20 standard amino acids.</p><p><sup>c</sup> The identity of the amino acid in the particular peptide that substituted the residue at the position under investigation.</p><p><sup>d</sup> All peptides other than those in the opposite column were not cleaved by SARS-CoV 3CL<sup>pro</sup>.</p><p><sup>e</sup> All peptides other than those in the opposite column were cleaved by SARS-CoV 3CL<sup>pro</sup>.</p><p><sup>f</sup> All 20 peptides were cleaved by SARS-CoV 3CL<sup>pro</sup>.</p></fn></table-wrap-foot></table-wrap><fig position="float" id="f2"><label>Figure 2</label><caption><p>Secondary screening: Mass spectrometry analysis of SARS-CoV 3CL<sup>pro</sup> cleavage products. The monoisotopic peaks of each peptide can be clearly shown. (<italic>A</italic>) PS02–1 peptide batch. The four peaks of peptides with Pro (P; <italic>m</italic>/<italic>z</italic>=1722.90), Cys (C; <italic>m</italic>/<italic>z</italic>=1728.86), Leu (L; <italic>m</italic>/<italic>z</italic>=1739.03), and Glu (E; <italic>m</italic>/<italic>z</italic>=1754.89) substitutions were resolved on the mass spectrum before cleavage assay (<italic>A-1</italic>); only the peptide peaks corresponding to Leu (*) substitutions were absent, whereas all other peaks remained after the reaction (<italic>A-2</italic>). (B) PS02–2 peptide batch. The three peaks of peptides with Val (V; <italic>m</italic>/<italic>z</italic>=1724.92), Asn (N; m/ z=1739.89), and Lys (K; <italic>m</italic>/<italic>z</italic>=1753.95) substitutions were resolved on the mass spectrum before cleavage assay (<italic>B-1</italic>), which were all remaining after the reaction (<italic>B-2</italic>). (<italic>C</italic>) PS02–3 peptide batch. The four peaks of peptides with Thr (T; <italic>m</italic>/<italic>z</italic>=1726.90), Ile (I; <italic>m</italic>/<italic>z</italic>=1739.03), Asp (D; <italic>m</italic>/<italic>z</italic>=1740.88), and Gln (Q; <italic>m</italic>/<italic>z</italic>=1753.91) substitutions were resolved on the mass spectrum before cleavage assay (<italic>C-1</italic>), which all were remaining after the reaction (<italic>C-2</italic>). The 1740.88 monoisotopic peak with high intensity (*) belongs to the mother peak of the peptide with Asp (D) substitution. (<italic>D</italic>) PS06-I peptide batch. The peak of peptide with Ile (I; <italic>m</italic>/<italic>z</italic>=1795.09) substitution was resolved on the mass spectrum before cleavage assay (<italic>D-1</italic>), which remained after the reaction (<italic>D-2</italic>). (<italic>E</italic>) PS06-L peptide batch. The peak of peptide with Leu (L; <italic>m</italic>/<italic>z</italic>=1795.09) substitution was resolved on the mass spectrum before cleavage assay (<italic>E-1</italic>); which remained after the reaction (<italic>E-2</italic>).</p></caption><graphic xlink:href="699fig2"></graphic></fig><table-wrap position="float" id="t3"><label>Table 3</label><caption><p>Summary of results from secondary screening</p></caption><graphic xlink:href="699tbl3"></graphic><table-wrap-foot><fn><p><sup>a</sup> Pro (<italic>m</italic>/<italic>z</italic> value of 1722.90) and Val (<italic>m</italic>/<italic>z</italic> value of 1724.92) substitutions with close masses were separated into PS02–1 and PS02–2 peptide batches, respectively.</p><p><sup>b</sup> Cys (<italic>m</italic>/<italic>z</italic> value of 1728.86) and Thr (<italic>m</italic>/<italic>z</italic> value of 1726.90) substitutions with close masses were separated into PS02–1 and PS02–3 peptide batches, respectively.</p><p><sup>c</sup> Leu (<italic>m</italic>/<italic>z</italic> value of 1739.03), Asn (<italic>m</italic>/<italic>z</italic> value of 1739.89), and Ile (<italic>m</italic>/<italic>z</italic> value of 1739.03) substitutions with close masses were separated into PS02–1, PS02–2, and PS02–3 peptide batches, respectively.</p><p><sup>d</sup> Glu (<italic>m</italic>/<italic>z</italic> value of 1754.89), Lys (<italic>m</italic>/<italic>z</italic> value of 1753.95), and Gln (<italic>m</italic>/<italic>z</italic> value of 1753.91) substitutions with close masses were separated into PS02–1, PS02–2, and PS02–3 peptide batches, respectively.</p><p><sup>e</sup> Asp (<italic>m</italic>/<italic>z</italic> value of 1740.88) and Asn (<italic>m</italic>/<italic>z</italic> value of 1739.89) substitutions with close masses were separated into PS02–2 and PS02–3 peptide batches, respectively.</p><p><sup>f</sup> Ile and Leu substitutions with identical masses (<italic>m</italic>/<italic>z</italic> value of 1795.09) were separated into PS06-I and PS06-L peptide batches, respectively.</p></fn></table-wrap-foot></table-wrap><fig position="float" id="f3"><label>Figure 3</label><caption><p>Interaction between SARS-CoV 3CL<sup>pro</sup> and PS01 control peptide as predicted by molecular docking. Overview (<italic>left</italic>) of the interaction between the SARS-CoV 3CL<sup>pro</sup> (purple) and peptide substrate PS01 (red) and the zoom-in view (<italic>right</italic>) of the conserved Gln residue (gray) in the P1 position at the cleavage site of the PS01 control peptide being docked into the catalytic dyad of SARS-CoV 3CL<sup>pro</sup>, which is composed of the Cys145 (orange) and His41 (yellow) residues.</p></caption><graphic xlink:href="699fig3"></graphic></fig><fig position="float" id="f4"><label>Figure 4</label><caption><p>Comparison between the molecular models of different selected peptide substrates docked to the active site of SARS-CoV 3CL<sup>pro</sup>. The Cys145 (orange) and His41 (yellow) residues in the catalytic dyad of SARS-CoV 3CL<sup>pro</sup> (purple) and the conserved Gln residue (gray) in the P1 position at the cleavage site of the peptide substrate (red) were shown. (<italic>A</italic>) PS02 peptide with Phe (green) in the P2 position was in close proximity to the active site of SARS-CoV 3CL<sup>pro</sup>. Peptide substrates that were unfavorable for SARS-CoV 3CL<sup>pro</sup> cleavage were deviated away from the active site of SARS-CoV 3CL<sup>pro</sup>, which include PS03 peptide with Pro (blue) in the P3 position (<italic>B</italic>); PS05 peptide with Pro (blue) in the P1′ position (<italic>C</italic>); PS05 peptide with Asp (blue) in the P1′ position that was in close proximity to the acidic residues (brown) at the active site (<italic>D</italic>); PS05 peptide with Glu (blue) in the P1′ position that was in close proximity to the acidic residues (brown) at the active site (<italic>E</italic>); PS06 peptide with Pro (blue) in the P2′ position (<italic>F</italic>); PS06 peptide with Ile (blue) in the P2′ position (<italic>G</italic>); PS06 peptide with Leu (blue) in the P2′ position (<italic>H</italic>).</p></caption><graphic xlink:href="699fig4"></graphic></fig></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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