Nuclear Magnetic Resonance Study of the Protolysis of Trimethylammonium Ion in Aqueous Solution—Order of the Reaction with Respect to Solvent

Luz, Z.; Meiboom, S.

doi:10.1063/1.1734254

Cited by 527 publications

(399 citation statements)

References 12 publications

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“…To explore this further, the T 2 was measured using a CPMG sequence with delays ranging from 0.2 to 2 ms. If exchange occurs on this time scale, then the measurement of T 2 should vary with the delay (16). As shown in Table 1 and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The T 2 measurements were made with a standard CPMG echo sequence (46), and the delay was set to 0.2, 0.5, 1, or 2 ms. If chemical exchange was present and the exchange rate is in the time scale of the delays, then one would expect the T 2 to vary with CPMG delay (16). Although the protein concentration was well below the dimerization dissociation constant (6 mM, 9), the monomeric form of the protein was verified in the NMR tube using diffusion measurements which employed pulsed field gradients (21).…”

Section: Methodsmentioning

confidence: 99%

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Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

Ahmed

Loh

Jane

et al. 2007

Journal of Biological Chemistry

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Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. Although the structure of the GluR2 binding domain (S1S2) is well known (agonist binding site between two lobes), little is known about the time scales of conformational transitions or the relationship between dynamics and function.19 F NMR ( 19 F-labeled tryptophan) spectroscopy was used to monitor motions in the S1S2 domain bound to ligands with varying efficacy and in the apo state. One tryptophan (Trp-671) undergoes chemical exchange in some but not all agonists, consistent with s-ms motion. The dynamics can be correlated to ligand affinity, and a likely source of the motion is a peptide bond capable of transiently forming hydrogen bonds across the lobe interface. Another tryptophan (Trp-767) appears to monitor motions of the relative positions of the lobes and suggests that the relative orientation in the apo-and antagonist-bound forms can exchange between at least two conformations on the ms time scale. Ionotropic glutamate receptors (GluRs)2 mediate the majority of excitatory synaptic transmission in the central nervous system of higher vertebrates (1) and play important roles in the formation of synaptic plasticity underlying higher order processes such as learning and memory as well as in neuronal development (2). In addition, ionotropic GluRs have been implicated in various neurodegenerative disorders such as Parkinson and Alzheimer diseases, Huntington chorea, and neurologic disorders including epilepsy and ischemic brain damage. Antagonists of glutamate receptors have been shown to limit tumor growth in a variety of human tumors and to inhibit tumor cell migration (3). In recent years many advances in characterizing the relationship between ionotropic GluR structure and function have been made. Ionotropic GluRs are membrane-bound receptor ion channels composed of multiple subunits arranged as a rosette, forming a central ion channel in which each subunit contributes to pore formation. Individual subunits are categorized by pharmacological properties, sequence, functionality, and biological roles into those that are sensitive 1) to the synthetic agonist N-methyl-D-aspartic acid (NR1, NR2A-D, NR3A-B), 2) to the synthetic agonist ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; GluR1-4), and 3) to the naturally occurring neurotoxin kainate (GluR5-7, KA1,2).The structural analysis of glutamate receptors was dramatically advanced by the finding that the extracellular agonist binding domain (S1S2 domain) could be expressed in isolation as a soluble protein (4) and by the subsequent solution of the crystal structure bound to kainate (5). As predicted by homology models (6), the S1S2 domain was found to be a bilobed structure with the agonist binding pocket located between the two lobes. The first glimpse of the structural basis of channel activation was the finding by Armstrong and Gouaux (7) that the apo state exhibited a considerably larger angle between the two lobes than the full agonist-bound...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

Ahmed

Loh

Jane

et al. 2007

Journal of Biological Chemistry

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“…Data for which R 2 changed by Ͻ2 s Ϫ1 over the entire range of cp were excluded from further analysis. The remaining residues were tested to determine whether the variation in R 2 was statistically significant by fitting each to a horizontal line and to a simplified two-site expression valid for fast exchange (25):…”

Section: Methodsmentioning

confidence: 99%

Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

McElheny

Schnell²,

Lansing³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

153

229

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Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP ؉ cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30°C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.enzyme catalysis ͉ hydride transfer ͉ NMR relaxation P roteins are dynamic molecular machines, and conformational fluctuations on a wide range of time scales are intimately associated with protein function. It has long been recognized that dynamic processes play an important role in the catalytic function of enzymes (1, 2). Protein motion is implicated in events such as binding of substrate or cofactor, allosteric regulation, and product release, and the catalyzed reaction itself is inherently dynamic, with changes in atomic positions occurring along the reaction coordinate (3). Despite mounting experimental evidence that the active sites of enzymes are inherently flexible (4-6), a detailed understanding of the relationship between the dynamics and thermodynamics of protein fluctuations and the catalytic process is currently lacking.A number of experimental and theoretical investigations have suggested that protein motions play an important role in catalysis by the enzyme dihydrofolate reductase (DHFR) (see refs. 7 and 8 for recent reviews). DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) through stereo-specific hydride transfer from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor. The enzyme is essential for tetrahydrofolate (THF) biosynthesis, plays a central role in promoting cell growth and proliferation, and is the target of several anticancer and antibiotic drugs. Escherichia coli DHFR has been subjected to extensive kinetic and structural studies that have defined the complete kinetic mechanism (9) and the structural transitions involved in the catalytic cycle (10, 11). During the reaction cycle (Fig. 1), the enzyme progresses through conformations in which the active site loops are in a closed state, in the holoenzyme and the Michaelis complex, and a series of product complexes in which the loops adopt an occluded conformation. In the closed state, the Met-20 loop (residues 9-24) packs against the nicotinamide ring of the cofactor and seals the active site (10). In the occluded state, Met-16 and Glu-17 in the Met-20 loop project into the active site and sterically occlude the binding site for the nicotinamide-r...

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“…While the contribution of the exchange with chemically shifted protons to T 2 is eliminated by CPMG with short t cp (25), the contribution of the residual dipolar interaction is not. This contribution can be eliminated by the application of the q-CPMG [908 À (t cp À 908 À t cp ) n À Acq] pulse sequence (26,27), with t cp short relative to the water proton exchange rate.…”

Section: The Nature Of 1 H T 2 Decay In Articular Cartilagementioning

confidence: 97%

Multinuclear NMR and microscopic MRI studies of the articular cartilage nanostructure

Shinar

Navon

2006

NMR in Biomedicine

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Studies of the structure of articular cartilage by a number of NMR spectroscopic and imaging techniques are reviewed. Advantage is taken of the fact that the NMR investigations can be done non-invasively on the intact tissue and do not require sectioning, slicing and decalcification as in the case of electron microscopy. The different contributions to 1 H T 2 relaxation are described and it is pointed out that ignoring the biexponential behavior of the transverse relaxation can lead to serious errors in the proton density measurements and the T 2 characterization of the articular cartilage. A way to slow the transverse relaxation and to minimize its angular dependence by the use of dipolar echo is described. 2 H double quantum filtered spectroscopic MRI is a powerful technique to follow the orientation and density of the collagen fibers in articular cartilage. Using this technique, it was found that attachment of the cartilage to the bone has a stabilizing effect on the collagen matrix and that the hydroxyapatite in the calcified zone resides near the collagen fibers but does not contribute to their order. In response to mechanical pressure, it was shown that the collagen fibers flatten near the surface and become crimped near the bone. A number of NMR techniques have been described for the measurement of 23 Na residual quadrupolar interaction. It was found that this can serve as a very sensitive measure of the depletion of proteoglycans. Finally, a combination of the above techniques was used to study a maturation of articular cartilage in pigs. The increased order and density of the collagen fibers from newborn to adult pigs revealed itself as a shortening of T 2 and significant increase of the residual quadrupolar interaction of both 2 H and 23 Na nuclei.

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Nuclear Magnetic Resonance Study of the Protolysis of Trimethylammonium Ion in Aqueous Solution—Order of the Reaction with Respect to Solvent

Cited by 527 publications

References 12 publications

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

Multinuclear NMR and microscopic MRI studies of the articular cartilage nanostructure

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