The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris.
Identifieur interne : 002629 ( PubMed/Corpus ); précédent : 002628; suivant : 002630The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris.
Auteurs : A R Kuchumov ; J C Taveau ; J N Lamy ; J S Wall ; R E Weber ; S N VinogradovSource :
- Journal of molecular biology [ 0022-2836 ] ; 1999.
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Hemoglobins, Oxygen.
- chemical , physiology : Blood Proteins.
- Animals, Kinetics, Oligochaeta.
Abstract
The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.
DOI: 10.1006/jmbi.1999.2825
PubMed: 10373372
Links to Exploration step
pubmed:10373372Le document en format XML
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The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">10373372</PMID><DateCompleted><Year>1999</Year><Month>07</Month><Day>28</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0022-2836</ISSN><JournalIssue CitedMedium="Print"><Volume>289</Volume><Issue>5</Issue><PubDate><Year>1999</Year><Month>Jun</Month><Day>25</Day></PubDate></JournalIssue><Title>Journal of molecular biology</Title><ISOAbbreviation>J. Mol. Biol.</ISOAbbreviation></Journal><ArticleTitle>The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris.</ArticleTitle><Pagination><MedlinePgn>1361-74</MedlinePgn></Pagination><Abstract><AbstractText>The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.</AbstractText><CopyrightInformation>Copyright 1999 Academic Press.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kuchumov</LastName><ForeName>A R</ForeName><Initials>AR</Initials><AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, 48201, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Taveau</LastName><ForeName>J C</ForeName><Initials>JC</Initials></Author><Author ValidYN="Y"><LastName>Lamy</LastName><ForeName>J N</ForeName><Initials>JN</Initials></Author><Author ValidYN="Y"><LastName>Wall</LastName><ForeName>J S</ForeName><Initials>JS</Initials></Author><Author ValidYN="Y"><LastName>Weber</LastName><ForeName>R E</ForeName><Initials>RE</Initials></Author><Author ValidYN="Y"><LastName>Vinogradov</LastName><ForeName>S N</ForeName><Initials>SN</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>J Mol Biol</MedlineTA><NlmUniqueID>2985088R</NlmUniqueID><ISSNLinking>0022-2836</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D001798">Blood Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D006454">Hemoglobins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C081784">hemoglobin linker chain L1 protein, Lumbricus terrestris</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C086641">hemoglobin linker chain L2, Neanthes diversicolor</NameOfSubstance></Chemical><Chemical><RegistryNumber>S88TT14065</RegistryNumber><NameOfSubstance UI="D010100">Oxygen</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001798" MajorTopicYN="N">Blood Proteins</DescriptorName><QualifierName UI="Q000502" MajorTopicYN="Y">physiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006454" MajorTopicYN="N">Hemoglobins</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009835" MajorTopicYN="Y">Oligochaeta</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010100" MajorTopicYN="N">Oxygen</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1999</Year><Month>6</Month><Day>22</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1999</Year><Month>6</Month><Day>22</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1999</Year><Month>6</Month><Day>22</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">10373372</ArticleId><ArticleId IdType="doi">10.1006/jmbi.1999.2825</ArticleId><ArticleId IdType="pii">S0022-2836(99)92825-5</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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