NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins

JOURNAL OF MOLECULAR BIOLOGY

卷:431

Article

Chalmers, Gordon R.¹  Eletsky, Alexander¹  Morris, Laura C.¹  Yang, Jeong-Yeh¹  Tian, Fang^1,2  Woods, Robert J.¹  Moremen, Kelley W.¹  Prestegard, James H.¹ 

[1] Univ Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA

[2] Penn State Coll Med, Biochem & Mol Biol, Hershey, PA USA

关键词: mammalian cell culture;    molecular dynamics;    ligand docking;    genetic algorithm;    sialyltransferase;   

DOI  :  10.1016/j.jmb.2019.04.029

来源: Elsevier

【 摘 要 】

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 N-15 or C-13, 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 lumina! domain of the sialyltransferase, rST6Ga11, in which all phenylalanines are labeled with N-15, 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. (C) 2019 Elsevier Ltd. All rights reserved.

【 授权许可】

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