NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins. Issue 12 (31st May 2019)

• Record Type: Journal Article
• Authors: Chalmers, Gordon R.; Eletsky, Alexander; Morris, Laura C.; Yang, Jeong-Yeh; Tian, Fang; Woods, Robert J.; Moremen, Kelley W.; Prestegard, James H.
• Is Part Of: Journal of molecular biology. Volume 431:Issue 12(2019)
• Page Start: 2369
• Page End: 2382
• Publication Date: 2019-05-31
• Subjects: mammalian cell culture -- molecular dynamics -- ligand docking -- genetic algorithm -- sialyltransferase; RDC residual dipolar coupling -- NOE nuclear Overhauser effect -- HSQC heteronuclear single quantum coherence -- MD molecular dynamics -- PRE paramagnetic relaxation enhancement -- WT wild type -- CMP cytidine monophosphate; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2019.04.029
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with 15 N or 13 C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with 15 N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells. Graphical abstract: Unlabelled Image Highlights: A software package for NMR resonance assignment in sparsely labeled proteins is described. NMR resonances in a sparsely labeled, mammalian-cell-expressed, protein are assigned. Paramagnetic effects on assigned resonances facilitate docking of an enzyme substrate.