Direct Measurements of the Dissociation-Rate Constant for Inhibitor-Enzyme Complexes via the T1ρ and T2 (CPMG) Methods

Davis, Donald G.; Perlman, Michael E.; London, Robert E.

doi:10.1006/jmrb.1994.1084

Cited by 283 publications

(284 citation statements)

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“…Residues for which an F test at the 99.9% confidence limit indicated that the dispersion was not significant were also excluded from further analysis. For each residue that displays statistically significant dispersion, the R 2 relaxation dispersion data at the two fields were fit simultaneously to the general equation for exchange between two sites, A and B (21,26):…”

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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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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“…Analysis of Exchange Data-The data from the CPMG experiments for Trp-671 were analyzed using the general expression for the transverse relaxation rate for two-site exchange, which is applicable to all chemical exchange time scales (47)(48)(49).…”

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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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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“…Slower ( s-ms) motions can thus be characterized either by relaxation dispersion [30][31][32] or the power dependence of T 1 . 33,34 …”

Section: Nmr Studies Of Nucleic Acids Dynamicsmentioning

confidence: 99%

“…33,34 This effect can be taken into account in a straightforward manner by correcting the measured T 1 according to:…”

Section: Nmr Studies Of Fast Molecular Motions By Relaxation Rate Meamentioning

confidence: 99%

NMR studies of dynamics in RNA and DNA by ¹³C relaxation

Shajani

Varani

2006

Biopolymers

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T he many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins, or other nucleic acids. 1,2 Although structurally more conservative and therefore dynamically less diverse, local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. 3 These statements make a compelling case to study the sequence dependent dynamics in nucleic acids; yet, residue-specific studies of nucleic acid dynamics are relatively few. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention, this spectroscopy being the only way to access information on a residue-by-residue basis. The questions that are being asked are how motion contributes to the affinity and specificity of biological interactions and what trajectories lead to the remarkably large conformational changes that are sometimes observed.RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. Figure 1 summarizes the NMR spectroscopic techniques commonly used to obtain information in each motional regime. Dynamics occurring on time scales that are considered ''fast'' in NMR spectroscopic studies (ps-ns) largely reflects bond librations associated with small amplitude and localized motions.

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Direct Measurements of the Dissociation-Rate Constant for Inhibitor-Enzyme Complexes via the T1ρ and T2 (CPMG) Methods

Cited by 283 publications

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

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

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

NMR studies of dynamics in RNA and DNA by ¹³C relaxation

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Direct Measurements of the Dissociation-Rate Constant for Inhibitor-Enzyme Complexes via the T1ρ and T2 (CPMG) Methods

Cited by 283 publications

References 0 publications

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

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

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

NMR studies of dynamics in RNA and DNA by 13C relaxation

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NMR studies of dynamics in RNA and DNA by ¹³C relaxation