tailieunhanh - Báo cáo khoa học: Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

AnEscherichia 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 tyro-sine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15 N-HSQC NMR spectra. | iFEBS Journal Translational incorporation of L-3 4-dihydroxyphenylalanine into proteins Kiyoshi Ozawa1 Madeleine J. Headlam1 Dmitri Mouradov2 Stephen J. Watt3 Jennifer L. Beck3 Kenneth J. Rodgers4 Roger T. Dean5 Thomas Huber2 Gottfried Otting1 and Nicholas E. Dixon1 1 Research Schoolof Chemistry Australian National university Canberra Australia 2 Departments of Biochemistry and Mathematics University of Queensland St. Lucia Australia 3 Department of Chemistry University of Wollongong Australia 4 The Heart Research Institute Sydney Australia 5 The University of Canberra Australia Keywords cell-free protein synthesis DOPA protein oxidation protein NMR protein misfolding Correspondence N. E. Dixon Research Schoolof Chemistry Australian NationalUniversity Canberra ACT 0200 Australia Fax 61 2 612 50750 Tel 61 2 612 54391 E-mail dixon@ Received 16 February 2005 revised 2 April 2005 accepted 25 April 2005 doi 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 .