title: Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET.
abstract: The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Kaihang
authors.initials: K
authors.lastname: Wang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Amit
authors.initials: A
authors.lastname: Sachdeva
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Cox
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Nabil M
authors.initials: NM
authors.lastname: Wilf
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Wallace
authors.affiliation: Oregon State University, Department of Biochemistry and Biophysics, 2011 ALS, Corvallis, OR97331.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D003062:Codon; D011506:Proteins; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine
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