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ABSTRACT: The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K(d) 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.
Protein Science 09/2010; 19(9):1673-85. · 2.80 Impact Factor
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ABSTRACT: Glycans that are either N-linked to asparagine or O-linked to serine or threonine are the hallmark of glycoproteins, a class of protein that dominates the mammalian proteome. These glycans perform important functions in cells and in some cases are required for protein activity. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying glycan structure and interactions, particularly in a form that exploits heteronuclei such as 13C. Here an approach is presented that that uses alpha-2,6-sialyltransferase (ST6Gal-I) to enzymatically add 13C-N-acetylneuraminic acid (NeuAc or sialic acid) to glycoproteins after their preparation using nonbacterial hosts. ST6Gal-I is itself a glycoprotein, and in this initial application, labeling of its own glycans and observation of these glycans by NMR are illustrated. The catalytic domain from rat ST6Gal-I was expressed in mammalian HEK293 cells. The glycans from the two glycosylation sites were analyzed with mass spectrometry and found to contain sialylated biantennary structures. The isotopic labeling approach involved removal of the native NeuAc residues from ST6Gal-I with neuraminidase, separation of the neuramindase with a lectin affinity column, and addition of synthesized 13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various 13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating 1H-13C3-13C2 atoms of the sugar ring.
Journal of the American Chemical Society 10/2008; 130(36):11864-5. · 9.91 Impact Factor
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ABSTRACT: N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme involved in the biosynthesis of asparagine-linked oligosaccharides. It is responsible for the transfer of N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor, uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), to the 6 position of the alpha-1-6 linked Man residue in N-linked oligosaccharide core structures. GnT-V up-regulation has been linked to increased cancer invasiveness and metastasis and, appropriately, targeted for drug development. However, drug design is impeded by the lack of structural information on the protein and the way in which substrates are bound. Even though the catalytic domain of this type II membrane protein can be expressed in mammalian cell culture, obtaining structural information has proved challenging due to the size of the catalytic domain (95 kDa) and its required glycosylation. Here, we present an experimental approach to obtaining information on structural characteristics of the active site of GnT-V through the investigation of the bound conformation and relative placement of its ligands, UDP-GlcNAc and beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-GlcpOOctyl. Nuclear magnetic resonance (NMR) spectroscopy experiments, inducing transferred nuclear Overhauser effect (trNOE) and saturation transfer difference (STD) experiments, were used to characterize the ligand conformation and ligand-protein contact surfaces. In addition, a novel paramagnetic relaxation enhancement experiment using a spin-labeled ligand analogue, 5'-diphospho-4-O-2,2,6,6-tetramethylpiperidine 1-oxyl (UDP-TEMPO), was used to characterize the relative orientation of the two bound ligands. The structural information obtained for the substrates in the active site of GnT-V can be useful in the design of inhibitors for GnT-V.
Journal of Molecular Biology 04/2007; 366(4):1266-81. · 4.00 Impact Factor
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ABSTRACT: Reductive 13C-methylation of proteins has been used as an isotope labeling strategy to study protein structure, function, and dynamics by nuclear magnetic resonance (NMR) spectroscopy. However, assigning the resulting 13C-dimethylamine peaks in a 1H-13C NMR spectrum has proved to be difficult, but it is important to expand the scope of the method. The assignment strategy presented here utilizes mass spectrometry (MS) for sequence identification and varying 13C/12C isotope ratios to correlate with NMR data. The site-specific reactivity of the lysines and N-terminal amine of a protein is exploited to produce a sample with varying 13C/12C ratios at each dimethylamine. MS and NMR are used to quantitate and correlate these ratios in order to assign peaks in the 1H-13C NMR spectrum. Hen egg white lysozyme was used as a model protein to demonstrate this assignment strategy.
Journal of the American Chemical Society 01/2006; 127(50):17626-7. · 9.91 Impact Factor
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ABSTRACT: A new difference probe for nuclear magnetic resonance (NMR) spectroscopy is presented. The difference probe uses two saddle-shaped coils to excite and detect two samples simultaneously. The samples are held in a specially modified 3-mm NMR tube with an Ultem plastic disk to separate the samples. The probe's resonant circuit contains two crossed diodes that passively switch the relative phase of each coil during the NMR experiment. The result is a difference spectrum from the two samples. The degree of cancellation of common signals was determined to be approximately 90%, and the application of the probe to relaxation-edited difference spectroscopy for identifying protein-ligand interactions was demonstrated using glutathione and glutathione S-transferase binding protein.
Analytical and Bioanalytical Chemistry 04/2004; 378(6):1520-7. · 3.78 Impact Factor
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ABSTRACT: An automated method for high-throughput nuclear magnetic resonance (NMR) spectroscopy has been developed using a four-coil Multiplex NMR probe. The probe is constructed with solenoidal microcoils optimized for detection of small volume, mass-limited samples and a flow-through design. Four samples can be simultaneously injected into the Multiplex probe with a robotics liquid handler and then analyzed in rapid succession using a selective excitation experiment. Due to the simultaneous injection of four samples and the reduced analysis time with rapid selective excitation, the analysis rate achieved thus far is as low as 1 sample/34 s for 1D 1H NMR.
Analytical Chemistry 11/2003; 75(19):5116-23. · 5.86 Impact Factor
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ABSTRACT: A unique probe designed to acquire nuclear magnetic resonance difference spectra of two samples is presented. The NMR Difference Probe contains two sample coils in a resonant circuit that switches between parallel excitation and serial acquisition to cancel common signals such as solvent peaks and impurities. Two samples containing a common analyte, acetonitrile, were used to demonstrate signal cancellation in a difference spectrum collected with a single pulse experiment. The cancellation was over 96% effective. The approach described has applications in the areas of solvent subtraction and spectral simplification.
Journal of Magnetic Resonance 06/2002; 156(1):97-103. · 2.14 Impact Factor
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12/1999;
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ABSTRACT: Samples of zeolites supported on optical microfibers were prepared with tetraethyl orthosilicate (TEOS) as a binding agent using sol-gel methods. A slower rate of TEOS hydrolysis in the sol-gel relative to the rate of silanol condensation was observed to be crucial for successful support of the zeolite particles on the optical fibers. The amount of water and the pH of the reaction gel were varied to control and optimize the rates of hydrolysis and condensation. Scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET) surface area measurements, and thermogravimetric analysis (TGA) were used to characterize the zeolite particles on the optical microfibers. The zeolite adhesion obtained was robust as the particles sustained spinning at 2000 Hz and suspension in flowing water for 12 h. The zeolite-supported optical fibers provide an improved catalyst system for the introduction of light into zeolite pores.
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Megan A Macnaughtan
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ABSTRACT: Advances in nuclear magnetic resonance (NMR) spectroscopy are often driven by the development of novel hardware and software. In this thesis, three new probes for high-throughput and difference spectroscopy are presented. The Multiplex NMR Probe is a four-coil NMR probe that can analyze four samples simultaneously and in rapid succession. An automated sample handling and analysis routine for high-throughput NMR was developed to simultaneously inject four samples into the Multiplex Probe and achieved an analysis rate of almost two samples per minute for 1D 1 H NMR. Two probes were developed for NMR difference spectroscopy. The Difference Probes have the ability to acquire a single, difference spectrum from two samples simultaneously. The dual-coil probes have a unique radiofrequency circuit that generates a phase difference between the two samples' NMR signals by switching the relative orientation of the radiofrequency transmitter and receiver during the NMR experiment. Crossed-diodes can be used as passive switches or single diodes can be used with an active bias circuit to create the necessary switching function during the NMR experiment. One Difference Probe was constructed with solenoidal-geometry micro detection coils and flow capabilities. Two samples containing a common analyte, were used to demonstrate signal cancellation in a difference spectrum collected with a single pulse experiment. The cancellation was over 96% effective. The approach described has applications in the areas of solvent subtraction, spectral simplification, and liquid chromatography (LC)-NMR. The second Difference Probe was constructed with saddle-shaped detection coils and uses a specially modified 3 mm NMR tube to hold the two samples. The degree of cancellation of common signals was determined to be approximately 90%, and the application of the probe to relaxation-edited difference spectroscopy for identifying protein-ligand interactions was demonstrated.
ETD Collection for Purdue University.
