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. 2007 Apr;14(4):409-18.
doi: 10.1016/j.chembiol.2007.02.010.

Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I

Shan Liu  1 , Andre Venot, Lu Meng, Fang Tian, Kelley W Moremen, Geert-Jan Boons, James H Prestegard
Affiliations

Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I

Shan Liu et al. Chem Biol. 2007 Apr.

Abstract

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.

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Figures

Figure 1
Figure 1
Structure of the Natural Donor CMP-NeuAc and Its Analog CMP-4CarboxyTEMPO
Figure 2
Figure 2. Scheme for the Synthesis of TEMPO Analogs of CMP-NeuAc
Key: (a) 30% H2SO4/NaCN, THF, < 15° C. (b) 27% HCl, H2O, 80° C. (c) CH2N2/Et2O, MeOH. (d) (iPr)2NP(Cl)OCH2CH2CN, Et(iPr)2N, CH2Cl2. (e) 1H-tetrazole, MeCN, –40° C. (f) t-BuOOH, MeCN. (g) DBU, THF. (h) NaOMe, MeOH-H2O.
Figure 3
Figure 3. 1H-15N HSQC Spectra of 15N-Phe ST6Gal I in the Presence of CMP-NeuAc Analogs
(A–D) Comparison of 1H-15N HSQC plots of (A) 15N-Phe ST6Gal I, (B) 15N-Phe ST6Gal I with a 10-fold molar excess of CMP-3FNeuAc, and (C and D) 15N-Phe ST6Gal I with a 4-fold excess of CMP-4carboxyTEMPO (C) before and (D) after reduction with a 5-fold excess of ascorbate (relative to the amount of CMP-4carboxyTEMPO). The crosspeaks experiencing changes due to binding dynamics and/or paramagnetic relaxation are boxed.
See this image and copyright information in PMC

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