Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins
Date
2005
Authors
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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Volume Title
Publisher
Blackwell Publishing Ltd
Abstract
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 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.
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Keywords
Keywords: 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
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Source
The FEBS Journal
Type
Journal article
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2037-12-31
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