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Translational incorporation of L -3,4-dihydroxyphenylalanine into proteins

Ozawa, Kiyoshi; Headlam, Madeleine; Mouradov, Dmitri; Watt, Stephen J; Beck, Jennifer; Rodgers, Kenneth; Dean, Roger; Dixon, Nicholas; Otting, Gottfried; Huber, Thomas

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An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and...[Show more]

dc.contributor.authorOzawa, Kiyoshi
dc.contributor.authorHeadlam, Madeleine
dc.contributor.authorMouradov, Dmitri
dc.contributor.authorWatt, Stephen J
dc.contributor.authorBeck, Jennifer
dc.contributor.authorRodgers, Kenneth
dc.contributor.authorDean, Roger
dc.contributor.authorDixon, Nicholas
dc.contributor.authorOtting, Gottfried
dc.contributor.authorHuber, Thomas
dc.date.accessioned2015-12-10T23:00:58Z
dc.identifier.issn1742-464X
dc.identifier.urihttp://hdl.handle.net/1885/61569
dc.description.abstractAn Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.
dc.publisherBlackwell Publishing Ltd
dc.sourceThe FEBS Journal
dc.subjectKeywords: DOPA; polypeptide; protein; tyrosine; amino acid substitution; article; cell free system; electrospray mass spectrometry; Escherichia coli; genetic transcription; heteronuclear single quantum coherence nitrogen nuclear magnetic resonance; nitrogen nuclear Cell-free protein synthesis; DOPA; Protein misfolding; Protein NMR; Protein oxidation
dc.titleTranslational incorporation of L -3,4-dihydroxyphenylalanine into proteins
dc.typeJournal article
local.description.notesImported from ARIES
local.identifier.citationvolume272
dc.date.issued2005
local.identifier.absfor060102 - Bioinformatics
local.identifier.ariespublicationU4217927xPUB621
local.type.statusPublished Version
local.contributor.affiliationOzawa, Kiyoshi, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationHeadlam, Madeleine, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationMouradov, Dmitri, University of Queensland
local.contributor.affiliationWatt, Stephen J, University of Wollongong
local.contributor.affiliationBeck, Jennifer, University of Wollongong
local.contributor.affiliationRodgers, Kenneth, University of Sydney
local.contributor.affiliationDean, Roger, University of Canberra
local.contributor.affiliationHuber, Thomas, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationOtting, Gottfried, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationDixon, Nicholas, College of Physical and Mathematical Sciences, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.issue12
local.bibliographicCitation.startpage3162
local.bibliographicCitation.lastpage3171
local.identifier.doi10.1111/j.1742-4658.2005.04735.x
dc.date.updated2015-12-10T08:25:36Z
local.identifier.scopusID2-s2.0-21344443051
CollectionsANU Research Publications

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