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

Authors

Kiyoshi Ozawa, University of WollongongFollow
Madeline J. Headlam, Australian National University
Dmitri Mouradov, University of Queensland
Stephen J. Watt, University of Wollongong
Jennifer L. Beck, University of WollongongFollow
Kenneth J. Rodgers, The Heart Research Institute
Roger T. Dean, University of Canberra
Thomas Huber, University of Queensland
Gottfried Otting, Australian National University
Nicholas E. Dixon, University of WollongongFollow

RIS ID

13158

Publication Details

Ozawa, K., Headlam, M., Mouradov, D., Watt, S., Beck, J. L., Rodgers, K., Dean, R., Huber, T., Otting, G. & Dixon, N. E. (2005). Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins. The FEBS Journal, 272 3162-3171.

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 clean ¹⁵N-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.