Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins.
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.
Ozawa
Kiyoshi
K
Research School of Chemistry, Australian National University, Canberra, Australia.
Headlam
Madeleine J
MJ
Mouradov
Dmitri
D
Watt
Stephen J
SJ
Beck
Jennifer L
JL
Rodgers
Kenneth J
KJ
Dean
Roger T
RT
Huber
Thomas
T
Otting
Gottfried
G
Dixon
Nicholas E
NE
D000595:Amino Acid Sequence; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D006639:Histidine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014443:Tyrosine / Q000378:metabolism
D010455:Peptides; D011506:Proteins; C081786:cyclophilin B; D014443:Tyrosine; D006639:Histidine; D004295:Dihydroxyphenylalanine; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase