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

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dc.contributor.authorOzawa, Kiyoshien_AU
dc.contributor.authorHeadlam, Madeleineen_AU
dc.contributor.authorMouradov, Dmitrien_AU
dc.contributor.authorWatt, Stephen Jen_AU
dc.contributor.authorBeck, Jenniferen_AU
dc.contributor.authorRodgers, Kennethen_AU
dc.contributor.authorDean, Rogeren_AU
dc.contributor.authorDixon, Nicholasen_AU
dc.contributor.authorOtting, Gottfrieden_AU
dc.contributor.authorHuber, Thomasen_AU
dc.date.accessioned2015-12-13T22:42:37Z
dc.date.issued2005
dc.date.updated2015-12-11T10:08:07Z
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.identifier.issn1742-464X
dc.identifier.urihttp://hdl.handle.net/1885/78854
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.bibliographicCitation.issue12
local.bibliographicCitation.lastpage3171
local.bibliographicCitation.startpage3162
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, University of Queensland
local.contributor.affiliationOtting, Gottfried, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationDixon, Nicholas, College of Physical and Mathematical Sciences, ANU
local.contributor.authoremailu4046684@anu.edu.au
local.contributor.authoruidOzawa, Kiyoshi, u4050581
local.contributor.authoruidHeadlam, Madeleine, u4051658
local.contributor.authoruidOtting, Gottfried, u4046684
local.contributor.authoruidDixon, Nicholas, u8102891
local.description.embargo2037-12-31
local.description.notesImported from ARIES
local.description.refereedYes
local.identifier.absfor100299 - Environmental Biotechnology not elsewhere classified
local.identifier.ariespublicationMigratedxPub7415
local.identifier.citationvolume272
local.identifier.doi10.1111/j.1742-4658.2005.04735.x
local.identifier.scopusID2-s2.0-21344443051
local.identifier.uidSubmittedByMigrated
local.type.statusPublished Version

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