Quantitative analysis of protein–ligand interactions by NMR

Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

doi:10.1016/j.pnmrs.2016.02.002

Cited by 94 publications

(88 citation statements)

References 97 publications

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“…Owing to the long nuclear spin decoherence time, characteristic time constants such as those governing chemical exchange can be determined under equilibrium up to the second time scale. 1,4,5 In an intermediate exchange regime, where the exchange rate is approximately equal to the difference in resonance frequencies of the two exchanged states ( k ex ≈ Δν ), a measurement of relaxation dispersion (RD) using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences yields information on minor molecular conformations that are not directly observable. 6–8 Kay and co-workers have applied the RD measurements to study a series of invisible or excited protein conformations.…”

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confidence: 99%

“…9–14 In addition to the conformational change of one macromolecule, binding interactions can also be quantitatively analyzed through the exchange between bound and free states using the RD method. 5,15,16 Binding mechanisms can then be distinguished by comparing intrinsic dissociation constants K D,i = k off / k on , which can be determined from RD, with apparent K D measured by other techniques. 5,16–19 In general, simple two-state, conformational selection and induced fit mechanisms follow K D,i = K D , K D,i < K D , and K D,i > K D respectively.…”

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confidence: 99%

“…5,15,16 Binding mechanisms can then be distinguished by comparing intrinsic dissociation constants K D,i = k off / k on , which can be determined from RD, with apparent K D measured by other techniques. 5,16–19 In general, simple two-state, conformational selection and induced fit mechanisms follow K D,i = K D , K D,i < K D , and K D,i > K D respectively. 5 In contrast to protein-detected RD measurements, recently developed ligand-detected RD experiments 17,20–22 do not require protein labeling, and protein concentrations can be low compared to ligand concentrations.…”

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confidence: 99%

“…5,16–19 In general, simple two-state, conformational selection and induced fit mechanisms follow K D,i = K D , K D,i < K D , and K D,i > K D respectively. 5 In contrast to protein-detected RD measurements, recently developed ligand-detected RD experiments 17,20–22 do not require protein labeling, and protein concentrations can be low compared to ligand concentrations. 13 C ligand-detected RD at natural abundance has been demonstrated to be viable.…”

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Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

2017

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Chemical exchange phenomena are ubiquitous in macromolecules, which undergo conformational change or ligand complexation. NMR relaxation dispersion (RD) spectroscopy based on a Carr-Purcell-Meiboom-Gill pulse sequence is widely applied to identify the exchange and measure the life time of intermediate states on the millisecond time scale. Advances in hyperpolarization methods improve the applicability of NMR spectroscopy when rapid acquisitions or low concentrations are required, through an increase in signal strength by several orders of magnitude. Here, we demonstrate the measurement of chemical exchange from a single aliquot of a ligand hyperpolarized by dissolution dynamic nuclear polarization (D-DNP). Transverse relaxation rates are measured simultaneously at different pulsing delays by dual-channel 19F NMR spectroscopy. This two-point measurement is shown to allow the determination of the exchange term in the relaxation rate expression. For the ligand 4-(trifluoromethyl)benzene-1-carboximidamide binding to the protein trypsin, the exchange term is found to be equal within error limits in neutral and acidic environments from D-DNP NMR spectroscopy, corresponding to a pre-equilibrium of trypsin deprotonation. This finding illustrates the capability for determination of binding mechanisms using D-DNP RD. Taking advantage of hyperpolarization, the ligand concentration in the exchange measurements can reach on the order of tens of μM and protein concentration can be below 1 μM, i.e. conditions typically accessible in drug discovery.

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Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

2017

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“…In protein titrations with ligands, NMR can be used to measure progressive shifting of peaks in the fast exchange regime 12 , which reflect population-weighted averages of the spectral contributions of the states in rapid equilibrium 2 . Principal component analysis (PCA) applied to 1 H and 15 N chemical shift changes can gain insight from the shifts in population due to pH, [ligand], or time 13–16 .…”

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Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts

Sarma

Wei

et al. 2017

Sci Rep

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Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates. We compared states the enzyme visits during its catalytic cycle. Collective responses of Pseudomonas PMM/PGM to phosphosugar substrates and inhibitor were assessed using NMR-detected titrations. Affinities were estimated from binding isotherms obtained by principal component analysis (PCA). Relationships among phosphosugar-enzyme associations emerge from PCA comparisons of the titrations. COordiNated Chemical Shifts bEhavior (CONCISE) analysis provides novel discrimination of three ligand-bound states of PMM/PGM harboring a mutation that suppresses activity. Enzyme phosphorylation and phosphosugar binding appear to drive the open dephosphorylated enzyme to the free phosphorylated state, and on toward ligand-closed states. Domain 4 appears central to collective responses to substrate and inhibitor binding. Hydrogen exchange reveals that binding of a substrate analogue enhances folding stability of the domains to a uniform level, establishing a globally unified structure. CONCISE and PCA of NMR spectra have discovered novel states of a well-studied enzyme and appear ready to discriminate other enzyme and ligand binding states.

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Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

2021

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The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution 19 F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.

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Quantitative analysis of protein–ligand interactions by NMR

Cited by 94 publications

References 97 publications

Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

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