title: Novel approaches in selective tryptophan isotope labeling by using Escherichia coli overexpression media.
abstract: NMR-based investigations of large protein complexes require optimized isotopic labeling schemes. We report new methods to introduce stable isotopes into tryptophan residues; these are fine-tuned to the requirements of the particular protein NMR experiment. Selective backbone labeling was performed by using a new α-ketoacid precursor as an additive in cell-based overexpression media. Additionally, we developed synthetic routes to certain isotopologues of indole with (13)C-(1)H spin systems surrounded by (12)C and (2)H. The corresponding proteins, overexpressed in the presence of these precursor compounds, can be effectively analyzed for conformational changes in tryptophan residues in response to external stimuli, such as interaction with other proteins or small molecules.
authors.affiliation: Institute of Organic Chemistry, University of Vienna, Währingerstrasse 38, 1090 Vienna (Austria).
authors.forename: Julia
authors.initials: J
authors.lastname: Schörghuber
authors.forename: Tomáš
authors.initials: T
authors.lastname: Sára
authors.forename: Marilena
authors.initials: M
authors.lastname: Bisaccia
authors.forename: Walther
authors.initials: W
authors.lastname: Schmid
authors.forename: Robert
authors.initials: R
authors.lastname: Konrat
authors.forename: Roman J
authors.initials: RJ
authors.lastname: Lichtenecker
mesh_terms: D002247:Carbon Isotopes; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D007211:Indoles; D011522:Protons; D014364:Tryptophan; D003903:Deuterium
keywords: NMR spectroscopy; indole; isotope labeling; protein expression; tryptophan