MERS‐CoV papain-like protease (PLpro): expression, purification, and spectroscopic/thermodynamic characterization
Identifieur interne : 000610 ( Pmc/Checkpoint ); précédent : 000609; suivant : 000611MERS‐CoV papain-like protease (PLpro): expression, purification, and spectroscopic/thermodynamic characterization
Auteurs : Ajamaluddin Malik [Arabie saoudite] ; Mohammad A. Alsenaidy [Arabie saoudite]Source :
- 3 Biotech [ 2190-572X ] ; 2017.
Abstract
Within a decade, MERS-CoV emerged with nearly four times higher case fatality rate than an earlier outbreak of SARS-CoV and spread out in 27 countries in short span of time. As an emerging virus, combating it requires an in-depth understanding of its molecular machinery. Therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. In this study, MERS-CoV papain-like protease (PLpro) was recombinantly expressed and purified. Thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (DMS) and thermal shift assay. DMS study showed that the PLpro undergoes a single thermal transition and follows a pathway of two-state folding with
Url:
DOI: 10.1007/s13205-017-0744-3
PubMed: 28560640
PubMed Central: 5449288
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">MERS‐CoV papain-like protease (PL<sup>pro</sup>): expression, purification, and spectroscopic/thermodynamic characterization</title><author><name sortKey="Malik, Ajamaluddin" sort="Malik, Ajamaluddin" uniqKey="Malik A" first="Ajamaluddin" last="Malik">Ajamaluddin Malik</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Department of Biochemistry, Protein Research Chair, College of Science,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2455, Riyadh, 11451 Saudi Arabia</nlm:aff><country xml:lang="fr">Arabie saoudite</country><wicri:regionArea>PO Box 2455, Riyadh</wicri:regionArea><wicri:noRegion>Riyadh</wicri:noRegion></affiliation></author><author><name sortKey="Alsenaidy, Mohammad A" sort="Alsenaidy, Mohammad A" uniqKey="Alsenaidy M" first="Mohammad A." last="Alsenaidy">Mohammad A. Alsenaidy</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Vaccines and Biologics Research Unit, Department of Pharmaceutics, College of Pharmacy,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2457, Riyadh, 11451 Saudi Arabia</nlm:aff><country xml:lang="fr">Arabie saoudite</country><wicri:regionArea>PO Box 2457, Riyadh</wicri:regionArea><wicri:noRegion>Riyadh</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28560640</idno><idno type="pmc">5449288</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449288</idno><idno type="RBID">PMC:5449288</idno><idno type="doi">10.1007/s13205-017-0744-3</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000260</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000260</idno><idno type="wicri:Area/Pmc/Curation">000260</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000260</idno><idno type="wicri:Area/Pmc/Checkpoint">000610</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000610</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">MERS‐CoV papain-like protease (PL<sup>pro</sup>): expression, purification, and spectroscopic/thermodynamic characterization</title><author><name sortKey="Malik, Ajamaluddin" sort="Malik, Ajamaluddin" uniqKey="Malik A" first="Ajamaluddin" last="Malik">Ajamaluddin Malik</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Department of Biochemistry, Protein Research Chair, College of Science,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2455, Riyadh, 11451 Saudi Arabia</nlm:aff><country xml:lang="fr">Arabie saoudite</country><wicri:regionArea>PO Box 2455, Riyadh</wicri:regionArea><wicri:noRegion>Riyadh</wicri:noRegion></affiliation></author><author><name sortKey="Alsenaidy, Mohammad A" sort="Alsenaidy, Mohammad A" uniqKey="Alsenaidy M" first="Mohammad A." last="Alsenaidy">Mohammad A. Alsenaidy</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Vaccines and Biologics Research Unit, Department of Pharmaceutics, College of Pharmacy,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2457, Riyadh, 11451 Saudi Arabia</nlm:aff><country xml:lang="fr">Arabie saoudite</country><wicri:regionArea>PO Box 2457, Riyadh</wicri:regionArea><wicri:noRegion>Riyadh</wicri:noRegion></affiliation></author></analytic><series><title level="j">3 Biotech</title><idno type="ISSN">2190-572X</idno><idno type="eISSN">2190-5738</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">Within a decade, MERS-CoV emerged with nearly four times higher case fatality rate than an earlier outbreak of SARS-CoV and spread out in 27 countries in short span of time. As an emerging virus, combating it requires an in-depth understanding of its molecular machinery. Therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. In this study, MERS-CoV papain-like protease (PL<sup>pro</sup>) was recombinantly expressed and purified. Thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (DMS) and thermal shift assay. DMS study showed that the PL<sup>pro</sup> undergoes a single thermal transition and follows a pathway of two-state folding with <italic>T</italic><sub>m</sub> and van’t Hoff enthalpy values of 54.4 ± 0.1 °C and 317.1 ± 3.9 kJ/mol, respectively. An orthogonal technique based on intrinsic tryptophan fluorescence also showed that MERS-CoV PL<sup>pro</sup> undergoes a single thermal transition and unfolds via a pathway of two-state folding with a <italic>T</italic><sub>m</sub> value of 51.4 °C. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">3 Biotech</journal-id><journal-id journal-id-type="iso-abbrev">3 Biotech</journal-id><journal-title-group><journal-title>3 Biotech</journal-title></journal-title-group><issn pub-type="ppub">2190-572X</issn><issn pub-type="epub">2190-5738</issn><publisher><publisher-name>Springer Berlin Heidelberg</publisher-name><publisher-loc>Berlin/Heidelberg</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28560640</article-id><article-id pub-id-type="pmc">5449288</article-id><article-id pub-id-type="publisher-id">744</article-id><article-id pub-id-type="doi">10.1007/s13205-017-0744-3</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>MERS‐CoV papain-like protease (PL<sup>pro</sup>): expression, purification, and spectroscopic/thermodynamic characterization</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Malik</surname><given-names>Ajamaluddin</given-names></name><address><phone>+966-114696241</phone><email>amalik@ksu.edu.sa</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Alsenaidy</surname><given-names>Mohammad A.</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Department of Biochemistry, Protein Research Chair, College of Science,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2455, Riyadh, 11451 Saudi Arabia</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 1773 5396</institution-id><institution-id institution-id-type="GRID">grid.56302.32</institution-id><institution>Vaccines and Biologics Research Unit, Department of Pharmaceutics, College of Pharmacy,</institution><institution>King Saud University,</institution></institution-wrap>PO Box 2457, Riyadh, 11451 Saudi Arabia</aff></contrib-group><pub-date pub-type="epub"><day>30</day><month>5</month><year>2017</year></pub-date><pub-date pub-type="pmc-release"><day>30</day><month>5</month><year>2017</year></pub-date><pub-date pub-type="ppub"><month>6</month><year>2017</year></pub-date><volume>7</volume><issue>2</issue><elocation-id>100</elocation-id><history><date date-type="received"><day>25</day><month>8</month><year>2016</year></date><date date-type="accepted"><day>6</day><month>2</month><year>2017</year></date></history><permissions><copyright-statement>© Springer-Verlag Berlin Heidelberg 2017</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><p id="Par1">Within a decade, MERS-CoV emerged with nearly four times higher case fatality rate than an earlier outbreak of SARS-CoV and spread out in 27 countries in short span of time. As an emerging virus, combating it requires an in-depth understanding of its molecular machinery. Therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. In this study, MERS-CoV papain-like protease (PL<sup>pro</sup>) was recombinantly expressed and purified. Thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (DMS) and thermal shift assay. DMS study showed that the PL<sup>pro</sup> undergoes a single thermal transition and follows a pathway of two-state folding with <italic>T</italic><sub>m</sub> and van’t Hoff enthalpy values of 54.4 ± 0.1 °C and 317.1 ± 3.9 kJ/mol, respectively. An orthogonal technique based on intrinsic tryptophan fluorescence also showed that MERS-CoV PL<sup>pro</sup> undergoes a single thermal transition and unfolds via a pathway of two-state folding with a <italic>T</italic><sub>m</sub> value of 51.4 °C. Our findings provide significant understandings of the thermodynamic and structural properties of MERS-CoV PL<sup>pro</sup>.</p></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Differential scanning fluorometry</kwd><kwd>Dynamic multimode spectroscopy</kwd><kwd>MERS</kwd><kwd>Papain-like protease</kwd><kwd>Thermal shift assay</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© King Abdulaziz City for Science and Technology 2017</meta-value></custom-meta></custom-meta-group></article-meta></front></pmc><affiliations><list><country><li>Arabie saoudite</li></country></list><tree><country name="Arabie saoudite"><noRegion><name sortKey="Malik, Ajamaluddin" sort="Malik, Ajamaluddin" uniqKey="Malik A" first="Ajamaluddin" last="Malik">Ajamaluddin Malik</name></noRegion><name sortKey="Alsenaidy, Mohammad A" sort="Alsenaidy, Mohammad A" uniqKey="Alsenaidy M" first="Mohammad A." last="Alsenaidy">Mohammad A. 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