Related ArticlesNMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins.
J Mol Biol. 2019 Apr 26;:
Authors: Chalmers GR, Eletsky A, Morris LC, Yang JY, Tian F, Woods RJ, Moremen KW, Prestegard JH
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 ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with 15N or 13C, 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 15N, 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.
PMID: 31034888 [PubMed - as supplied by publisher]
[NMR paper] NMR assignments of sparsely labeled proteins using a genetic algorithm.
NMR assignments of sparsely labeled proteins using a genetic algorithm.
Related Articles NMR assignments of sparsely labeled proteins using a genetic algorithm.
J Biomol NMR. 2017 Mar 13;:
Authors: Gao Q, Chalmers GR, Moremen KW, Prestegard JH
Abstract
Sparse isotopic labeling of proteins for NMR studies using single types of amino acid ((15)N or (13)C enriched) has several advantages. Resolution is enhanced by reducing numbers of resonances for large proteins, and isotopic labeling becomes economically feasible for glycoproteins...
nmrlearner
Journal club
0
03-16-2017 06:36 PM
NMR assignments of sparsely labeled proteins using a genetic algorithm
NMR assignments of sparsely labeled proteins using a genetic algorithm
Abstract
Sparse isotopic labeling of proteins for NMR studies using single types of amino acid (15N or 13C enriched) has several advantages. Resolution is enhanced by reducing numbers of resonances for large proteins, and isotopic labeling becomes economically feasible for glycoproteins that must be expressed in mammalian cells. However, without access to the traditional triple resonance strategies that require uniform isotopic labeling, NMR assignment of crosspeaks in...
nmrlearner
Journal club
0
03-14-2017 10:36 PM
Bacterial production of site specific 13 C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins
Bacterial production of site specific 13 C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins
Abstract
Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or...
nmrlearner
Journal club
0
12-27-2016 11:04 PM
[NMR paper] NMR Backbone Assignment of Large Proteins by Using (13) C? -Only Triple-Resonance Experiments.
NMR Backbone Assignment of Large Proteins by Using (13) C? -Only Triple-Resonance Experiments.
NMR Backbone Assignment of Large Proteins by Using (13) C? -Only Triple-Resonance Experiments.
Chemistry. 2016 Jun 8;
Authors: Wei Q, Chen J, Mi J, Zhang J, Ruan K, Wu J
Abstract
Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical-shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency...
nmrlearner
Journal club
0
06-09-2016 07:44 PM
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Abstract In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope...
nmrlearner
Journal club
0
01-09-2011 12:46 PM
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 4 January 2011</br>
Jie, Wen , Jihui, Wu , Pei, Zhou</br>
Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (H?, C?, C? and CO) for establishing sequential backbone assignment. Because most conventional 4-D...
nmrlearner
Journal club
0
01-05-2011 11:03 AM
[NMR paper] Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N a
Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II.
J Mol Biol. 1996 Dec 20;264(5):1101-16
Authors: Venters RA, Farmer BT, Fierke CA, Spicer LD
Perdeuteration of all non-exchangeable proton sites can...
NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins.
Linear Mode
