TY - JOUR
T1 - Spin-Labeled Analogs of CMP-NeuAc as NMR Probes of the α-2,6-Sialyltransferase ST6Gal I
AU - Liu, Shan
AU - Venot, Andre
AU - Meng, Lu
AU - Tian, Fang
AU - Moremen, Kelley W.
AU - Boons, Geert Jan
AU - Prestegard, James H.
N1 - Funding Information:
This work was supported by a grant from the National Center for Research Resources of the National Institutes of Health (NIH) (RR05351) in support of the Research Resource for Integrated Glycotechnology, by a research grant from the National Institute of General Medical Sciences (GM033225), and a grant from the National Institute of General Medicine of the NIH (GM066340) in support of the Southeast Collaboratory for High Field NMR. We thank Laura Morris for her help in ST6Gal I modeling.
PY - 2007/4/20
Y1 - 2007/4/20
N2 - Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
AB - Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
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U2 - 10.1016/j.chembiol.2007.02.010
DO - 10.1016/j.chembiol.2007.02.010
M3 - Article
C2 - 17462576
AN - SCOPUS:34247184796
SN - 1074-5521
VL - 14
SP - 409
EP - 418
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 4
ER -