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dc.contributor.authorGu, Zhenyu
dc.contributor.authorZitzewitz, Jill A.
dc.contributor.authorMatthews, C. Robert
dc.date2022-08-11T08:09:32.000
dc.date.accessioned2022-08-23T16:34:53Z
dc.date.available2022-08-23T16:34:53Z
dc.date.issued2007-03-16
dc.date.submitted2009-03-16
dc.identifier.citation<p>J Mol Biol. 2007 Apr 27;368(2):582-94. Epub 2007 Feb 20. <a href="http://dx.doi.org/10.1016/j.jmb.2007.02.027">Link to article on publisher's site</a></p>
dc.identifier.issn0022-2836 (Print)
dc.identifier.doi10.1016/j.jmb.2007.02.027
dc.identifier.pmid17359995
dc.identifier.urihttp://hdl.handle.net/20.500.14038/38434
dc.description.abstractTo test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=17359995&dopt=Abstract">Link to Article in PubMed</a></p>
dc.relation.urlhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2040069/
dc.subjectAmino Acid Motifs
dc.subjectAmino Acid Sequence
dc.subjectDeuterium
dc.subject*Deuterium Exchange Measurement
dc.subjectIndole-3-Glycerol-Phosphate Synthase
dc.subjectModels, Molecular
dc.subjectMolecular Sequence Data
dc.subjectMolecular Weight
dc.subjectPepsin A
dc.subjectPeptides
dc.subject*Protein Folding
dc.subjectProtein Structure, Secondary
dc.subjectProtons
dc.subject*Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
dc.subjectStructure-Activity Relationship
dc.subjectSulfolobus solfataricus
dc.subjectTriose-Phosphate Isomerase
dc.subjectUrea
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleMapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus
dc.typeJournal Article
dc.source.journaltitleJournal of molecular biology
dc.source.volume368
dc.source.issue2
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/1300
dc.identifier.contextkey782972
html.description.abstract<p>To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.</p>
dc.identifier.submissionpathoapubs/1300
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages582-94


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