Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride.
Identifieur interne : 001F68 ( PubMed/Corpus ); précédent : 001F67; suivant : 001F69Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride.
Auteurs : Hui-Ping Chang ; Chi-Yuan Chou ; Gu-Gang ChangSource :
- Biophysical journal [ 0006-3495 ] ; 2007.
English descriptors
- KwdEn :
- Binding Sites, Catalytic Domain, Circular Dichroism, Cysteine Endopeptidases (chemistry), Guanidine (chemistry), Indicators and Reagents, Models, Molecular, Protein Denaturation, Protein Folding, Protein Structure, Quaternary, Protein Structure, Tertiary, SARS Virus (enzymology), Viral Proteins (chemistry).
- MESH :
- chemical , chemistry : Cysteine Endopeptidases, Guanidine, Viral Proteins.
- enzymology : SARS Virus.
- Binding Sites, Catalytic Domain, Circular Dichroism, Indicators and Reagents, Models, Molecular, Protein Denaturation, Protein Folding, Protein Structure, Quaternary, Protein Structure, Tertiary.
Abstract
Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.
DOI: 10.1529/biophysj.106.091736
PubMed: 17142288
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pubmed:17142288Le document en format XML
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The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">17142288</PMID><DateCompleted><Year>2007</Year><Month>05</Month><Day>09</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>07</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0006-3495</ISSN><JournalIssue CitedMedium="Print"><Volume>92</Volume><Issue>4</Issue><PubDate><Year>2007</Year><Month>Feb</Month><Day>15</Day></PubDate></JournalIssue><Title>Biophysical journal</Title><ISOAbbreviation>Biophys. J.</ISOAbbreviation></Journal><ArticleTitle>Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride.</ArticleTitle><Pagination><MedlinePgn>1374-83</MedlinePgn></Pagination><Abstract><AbstractText>Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Chang</LastName><ForeName>Hui-Ping</ForeName><Initials>HP</Initials><AffiliationInfo><Affiliation>Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan. huiping_chp@hotmail.com</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chou</LastName><ForeName>Chi-Yuan</ForeName><Initials>CY</Initials></Author><Author ValidYN="Y"><LastName>Chang</LastName><ForeName>Gu-Gang</ForeName><Initials>GG</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2006</Year><Month>12</Month><Day>01</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biophys J</MedlineTA><NlmUniqueID>0370626</NlmUniqueID><ISSNLinking>0006-3495</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D007202">Indicators and Reagents</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014764">Viral Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="C099456">3C-like proteinase, Coronavirus</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="D003546">Cysteine Endopeptidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>JU58VJ6Y3B</RegistryNumber><NameOfSubstance UI="D019791">Guanidine</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><CommentsCorrectionsList><CommentsCorrections RefType="ErratumIn"><RefSource>Biophys J. 2007 Jul 15;93(2):704</RefSource></CommentsCorrections></CommentsCorrectionsList><MeshHeadingList><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020134" MajorTopicYN="N">Catalytic Domain</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002942" MajorTopicYN="N">Circular Dichroism</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003546" MajorTopicYN="N">Cysteine Endopeptidases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019791" MajorTopicYN="N">Guanidine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007202" MajorTopicYN="N">Indicators and Reagents</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008958" MajorTopicYN="Y">Models, Molecular</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011489" MajorTopicYN="N">Protein Denaturation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017510" MajorTopicYN="Y">Protein Folding</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020836" MajorTopicYN="N">Protein Structure, Quaternary</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017434" MajorTopicYN="N">Protein Structure, Tertiary</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014764" MajorTopicYN="N">Viral Proteins</DescriptorName><QualifierName UI="Q000737" 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