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ABSTRACT: Enzymatic addition of a N-acetyl-galactosamine (GalNAc) to isotopically labeled interferon alpha-2a (IFNα2a) produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-13C,15N-IFNα2a. The three dimensional structure of the resulting GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear overhauser enhancement measurements revealed that the addition of a single monosaccharide unit on Thr-106 significantly slows motions of the glycosylation loop in the nanosecond time scale. Subsequent addition of a galactose (Gal) unit produced Gal(β1,3)GalNAcα-13C,15N-IFNα2a. This extension resulted in a further decrease of the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: The stereochemistry of castor stearoyl-ACP Delta(9) desaturase-mediated 10-sulfoxidation has been determined. This was accomplished by (19)F NMR analysis of a fluorine-tagged product, 18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA. Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl substrate. These data validate the use of thia probes to determine the stereochemistry and cryptoregiochemistry of desaturase-mediated oxidations.
Organic & Biomolecular Chemistry 03/2010; 8(6):1322-8. · 3.70 Impact Factor
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ABSTRACT: This article reports the results of our investigation of the effects of pH and various formulations on the conformation of interferon (IFN) alpha-2a and IFN alpha-2b using the NMR fingerprinting assay. Samples of (15)N-IFN alpha-2 were produced and their activity was inferred by comparing their NMR spectra with those recorded for the corresponding European Directorate for the Quality of Medicines (EDQM) reference standards. The proteins were then mixed with appropriate excipients to reproduce formulations used in innovator products of Roferon-A and Intron-A and deformulated via cation-exchange chromatography. The conformation of IFN alpha-2 was monitored by two-dimensional (2D)-NMR spectroscopy at various pHs, after formulation and deformulation procedures. Our results show that the process does not alter the conformation of IFN alpha-2 and that the optimal pH for deformulation is 4.0 +/- 0.5. Variation in pH below 3.0 causes the protein to unfold, whereas above pH 4.5, the three-dimensional (3D) fold is maintained, but the NMR spectra indicate a propensity to oligomerize. This behaviour is reversible upon readjusting the pH to 3.5-4.5. Here, we demonstrate the applicability of NMR to assess the structure of protein therapeutics. The proposed method can assist in validating analytical methods that require deformulation of IFN-based products.
Journal of Pharmaceutical Sciences 02/2010; 99(8):3334-42. · 3.06 Impact Factor
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ABSTRACT: Chiral fluorine-tagged sulfoxides of known absolute configuration have been synthesized. These compounds are required as reference standards to validate a (19)F NMR-based micromethod for the stereochemical analysis of biosynthetic fatty acyl sulfoxides.
Bioorganic & medicinal chemistry letters 08/2009; 19(17):5146-50. · 2.65 Impact Factor
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ABSTRACT: Resistance of cancer cells to oncotherapeutics designed to trigger programmed cell death (a.k.a. apoptosis) greatly limits clinical efficacy. The human FLN29 protein may play a role in this process via protein-protein interactions. Here we report the NMR spectral assignment of the N-terminal TRAF2/6-RING-zinc finger-like domain of this protein.
Biomolecular NMR Assignments 07/2008; 2(1):33-6. · 0.72 Impact Factor
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ABSTRACT: The distribution of products obtained from stearoyl CoA Delta9 desaturase-mediated oxidation in Saccharomyces cerevisiae has been measured directly for the first time using an omega-fluorinated fatty acid substrate.
Organic & Biomolecular Chemistry 12/2005; 3(21):3979-83. · 3.70 Impact Factor
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ABSTRACT: The stereochemistry of fatty acid desaturase-mediated sulfoxidation can be evaluated at micromolar concentrations of analyte using (19)F NMR in combination with a chiral shift reagent: (S)-(+)-MPAA.
Bioorganic & Medicinal Chemistry Letters 07/2005; 15(11):2799-802. · 2.55 Impact Factor
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ABSTRACT: In this study, a series of novel luminescent cyclometalated Ir(III) complexes has been synthesized and evaluated for use in unimolecular oxygen-sensing materials. The complexes Ir(C6)(2)(vacac), 1, Ir(ppy)(2)(vacac), 2, fac-Ir(ppy)(2)(vppy), 3, and mer-Ir(ppy)(2)(vppy), 4, where C6 = Coumarin 6, vacac = allylacetoacetate, ppy = 2-phenylpyridine, and vppy = 2-(4-vinylphenyl)pyridine, all have pendent vinyl or allyl groups for polymer attachment via the hydrosilation reaction. These luminophore complexes were characterized by NMR, absorption, and emission spectroscopy, luminescence lifetime and quantum yield measurements, elemental analysis, and cyclic voltammetry. Complex 1 was structurally characterized using X-ray crystallography, and a series of 1-D ((1)H, (13)C) and 2-D ((1)H-(1)H, (1)H-(13)C) NMR experiments were used to resolve the solution structure of 4. Complexes 1 and 3 displayed the longest luminescence lifetimes and largest quantum efficiencies in solution (tau = 6.0 micros, phi = 0.22 for 1; tau = 0.4 micros, phi = 0.2 for 3) and, as result, are the most promising candidates for future luminescence-quenching-based oxygen-sensing studies.
Journal of the American Chemical Society 07/2004; 126(24):7619-26. · 9.91 Impact Factor
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ABSTRACT: A series of 18-fluoro thiastearates were prepared and incubated with a yeast Δ9-desaturating system. The relative efficiency of desaturase-mediated sulfoxidation was monitored via19F-NMR analysis of the sulfoxide products, and a strong preference for oxo transfer to the S-atom occupying the 9-position was confirmed. The oxidation profile obtained in this manner matched that of analogous experiments with non-fluorinated substrates. These results form the basis of a versatile 19F-NMR-based method for mapping the position of the putative diiron oxidant relative to substrate, and has potential application to the study of membrane-bound desaturases in vitro.
Helvetica Chimica Acta 11/2003; 86(11):3688 - 3697. · 1.48 Impact Factor
