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In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein

Williams, Neal; Prosselkov, Pavel; Liepinsh, Edvards; Line, Inara; Sharipo, Anatoly; Littler, Dene R; Curmi, Paul; Dixon, Nicholas; Otting, Gottfried

Description

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not...[Show more]

dc.contributor.authorWilliams, Neal
dc.contributor.authorProsselkov, Pavel
dc.contributor.authorLiepinsh, Edvards
dc.contributor.authorLine, Inara
dc.contributor.authorSharipo, Anatoly
dc.contributor.authorLittler, Dene R
dc.contributor.authorCurmi, Paul
dc.contributor.authorDixon, Nicholas
dc.contributor.authorOtting, Gottfried
dc.date.accessioned2015-12-13T22:25:10Z
dc.identifier.issn0021-9258
dc.identifier.urihttp://hdl.handle.net/1885/73110
dc.description.abstractA synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ∼ 14 °C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (ΔΔG ≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.
dc.publisherAmerican Society for Biochemistry and Molecular Biology Inc
dc.sourceJournal of Biological Chemistry
dc.subjectKeywords: Amino acids; Entropy; Escherichia coli; Free energy; Hydrogen bonds; Nuclear magnetic resonance spectroscopy; Thermodynamics; Protein cyclization; Proteins; bacterial protein; DNA B; intein; article; Cyanobacterium; cyclization; entropy; Escherichia coli;
dc.titleIn vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein
dc.typeJournal article
local.description.notesImported from ARIES
local.description.refereedYes
local.identifier.citationvolume277
dc.date.issued2002
local.identifier.absfor110106 - Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics)
local.identifier.ariespublicationMigratedxPub3559
local.type.statusPublished Version
local.contributor.affiliationWilliams, Neal, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationProsselkov, Pavel, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationLiepinsh, Edvards, Karolinska Institute
local.contributor.affiliationLine, Inara, University of Latvia
local.contributor.affiliationSharipo, Anatoly, University of Latvia
local.contributor.affiliationLittler, Dene R, University of New South Wales
local.contributor.affiliationCurmi, Paul, University of New South Wales
local.contributor.affiliationOtting, Gottfried, Karolinska Institute
local.contributor.affiliationDixon, Nicholas, College of Physical and Mathematical Sciences, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.issue10
local.bibliographicCitation.startpage7790
local.bibliographicCitation.lastpage7798
local.identifier.doi10.1074/jbc.M110303200
dc.date.updated2015-12-11T08:13:28Z
local.identifier.scopusID2-s2.0-0037040880
CollectionsANU Research Publications

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