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Translational incorporation of L ‐3,4‐dihydroxyphenylalanine into proteins
Author(s) -
Ozawa Kiyoshi,
Headlam Madeleine J.,
Mouradov Dmitri,
Watt Stephen J.,
Beck Jennifer L.,
Rodgers Kenneth J.,
Dean Roger T.,
Huber Thomas,
Otting Gottfried,
Dixon Nicholas E.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04735.x
Subject(s) - tyrosine , cell free protein synthesis , biochemistry , chemistry , dihydroxyphenylalanine , protein biosynthesis , protein aggregation , redox , intracellular , ribosome , translation (biology) , cell free system , cell , biophysics , biology , messenger rna , rna , in vitro , gene , dopamine , organic chemistry , neuroscience
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 clean 15 N‐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.