title: Site-selective13C labeling of proteins using erythrose.
abstract: NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with13C and/or1H, which is achieved in the most general way by using site-selectively13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively13C-enriched erythrose (1-, 2-, 3- and 4-13C) as a suitable precursor for13C labeled aromatic side chains. We quantify13C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the13C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated13C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective13C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.
authors.affiliation: Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P.O. Box 124, 22100, Lund, Sweden. ulrich.weininger@physik.uni-halle.de.|Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, 06120, Halle (Saale), Germany. ulrich.weininger@physik.uni-halle.de.
authors.forename: Ulrich
authors.identifier: 0000-0003-0841-8332
authors.initials: U
authors.lastname: Weininger
mesh_terms: D066241:Carbon-13 Magnetic Resonance Spectroscopy; D005947:Glucose / Q000737:chemistry; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry*; D013780:Tetroses / Q000737:chemistry*
chemical_list: D011506:Proteins; D013780:Tetroses; D005947:Glucose; C073321:erythrose
keywords: Aromatic side chain; Isotope labeling; Protein dynamics; Relaxation