Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I.
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 (15)N phenylalanine-labeled version of the structurally uncharacterized, alpha-2,6-linked sialyltransferase, ST6Gal I.
Liu
Shan
S
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Venot
Andre
A
Meng
Lu
L
Tian
Fang
F
Moremen
Kelley W
KW
Boons
Geert-Jan
GJ
Prestegard
James H
JH
D003497:Cyclic N-Oxides / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D012799:Sialyltransferases / Q000737:chemistry*; D013113:Spin Labels; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D003497:Cyclic N-Oxides; D015335:Molecular Probes; D009587:Nitrogen Isotopes; D013113:Spin Labels; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; C003959:TEMPO; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase