title: Selective (15)N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy.
abstract: The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective (15)N-labeling of side-chain amides, however, can be a challenge due to enzyme-catalyzed exchange of amide groups during protein synthesis. In the present study, we developed an efficient way of selectively labeling the side chains of asparagine, or asparagine and glutamine residues with (15)NH2. Using the biosynthesis pathway of tryptophan, a protocol was also established for simultaneous selective (15)N-labeling of the side-chain NH groups of asparagine, glutamine, and tryptophan. In combination with site-specific tagging of the target protein with a lanthanide ion, we show that selective detection of (15)N-labeled side-chains of asparagine and glutamine allows determination of magnetic susceptibility anisotropy tensors based exclusively on pseudocontact shifts of amide side-chain protons.
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Chan
authors.initials: C
authors.lastname: Cao
authors.forename: Jia-Liang
authors.initials: JL
authors.lastname: Chen
authors.forename: Yin
authors.initials: Y
authors.lastname: Yang
authors.forename: Feng
authors.initials: F
authors.lastname: Huang
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: Xun-Cheng
authors.initials: XC
authors.lastname: Su
mesh_terms: D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D005973:Glutamine; D001216:Asparagine