Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions (original) (raw)
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Zeitschrift für Naturforschung. C, Journal of biosciences
Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest con...
Zeitschrift für Naturforschung C, 2001
In this paper we present proton spin-lattice ( T1) and spin-spin ( T2) relaxation times measured vs. concentration, temperature, pulse interval (τCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (B SA ) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46% ). The functional dependence of the spin-spin relaxation time vs. τCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spinlattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exc...
Magnetic Resonance in Medicine, 2011
Chemical exchange saturation transfer (CEST) and spin-locking (SL) experiments were both able to probe the exchange process between protons of non-equivalent chemical environments. To compare the characteristics of the CEST and SL approaches in the study of chemical exchange effects, we performed CEST and SL experiments at varied pH and concentrated metabolites with exchangeable amide, amine, and hydroxyl protons at 9.4 T. Our results show that: i) On-resonance SL is most sensitive to chemical exchanges in the intermediate exchange regime and is able to detect hydroxyl and amine protons on a millimolar concentration scale. Off-resonance SL and CEST approaches are sensitive to slow-exchanging protons when an optimal SL or saturation pulse power matches the exchanging rate, respectively. ii) Offset frequency-dependent SL and CEST spectra are very similar, and can be explained well with an SL model recently developed by Trott and Palmer. iii) The exchange rate and population of metabolite protons can be determined from offset-dependent SL or CEST spectra or from on-resonance SL relaxation dispersion measurements. iv) The asymmetry of the magnetization transfer ratio (MTR asym ) is highly dependent on the choice of saturation pulse power. In the intermediate exchange regime, MTR asym becomes complicated and should be interpreted with care.
Journal of Biomolecular NMR, 2020
Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1 H-15 N 2D correlation experiments. Here we introduce 2D 13 C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarizationselective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).
Hyperpolarized Water to Study Protein–Ligand Interactions
The Journal of Physical Chemistry Letters, 2015
The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 μM so that one can verify in a few seconds whether the proteins remain intact or have been denatured
Magnetic Resonance in Medicine, 2012
Chemical exchange saturation transfer (CEST) and spin-locking (SL) experiments were both able to probe the exchange process between protons of non-equivalent chemical environments. To compare the characteristics of the CEST and SL approaches in the study of chemical exchange effects, we performed CEST and SL experiments at varied pH and concentrated metabolites with exchangeable amide, amine, and hydroxyl protons at 9.4 T. Our results show that: i) On-resonance SL is most sensitive to chemical exchanges in the intermediate exchange regime and is able to detect hydroxyl and amine protons on a millimolar concentration scale. Off-resonance SL and CEST approaches are sensitive to slow-exchanging protons when an optimal SL or saturation pulse power matches the exchanging rate, respectively. ii) Offset frequency-dependent SL and CEST spectra are very similar, and can be explained well with an SL model recently developed by Trott and Palmer. iii) The exchange rate and population of metabolite protons can be determined from offset-dependent SL or CEST spectra or from on-resonance SL relaxation dispersion measurements. iv) The asymmetry of the magnetization transfer ratio (MTR asym ) is highly dependent on the choice of saturation pulse power. In the intermediate exchange regime, MTR asym becomes complicated and should be interpreted with care.
New NMR methods for the characterization of bound waters in macromolecules
Journal of the American Chemical Society, 1993
The study of water molecules which are associated with macromolecules, either a t the surface or buried within the interior, is important because these waters play significant structural, catalytic, and/or recognition roles. We demonstrate new N M R techniques that will allow the determination of the lifetimes of certain bound water molecules. These techniques employ (i) pulsed field gradients (PFG) for diffusion editing, (ii) isotope editing for selective detection and solvent suppression, and (iii) selective excitation for efficiency of acquisition. Using a uniformly ISN-labeled fragment of the Escherichia coli chaperone protein, DnaJ, we show that a range of exchange lifetimes is observed for protons that physically exchange between water and amide sites. These methods will similarly allow the differentiation of bound water molecules on the basis of their lifetimes in the bound state and will be of general utility in the future for detailed studies of the dynamics of bound waters that have lifetimes in the 100 ps to 10 ms range.
Journal of Magnetic Resonance (1969), 1985
The study of molecules in dilute aqueous solution by 'H NMR has been hampered by the strong water resonance. Not only does the water resonance obscure a large part of the spectrum, it also makes it difficult to detect the much weaker resonances from the solute molecules by pulse Fourier transform NMR because of the limited dynamic range of analog-todigital converters. Several methods have been developed in attempts to solve the dynamic range problem, including selective saturation of the water resonance (1, 2), the inversion recovery pulse sequence (WEFT) (3, 4) and selective excitation pulse sequences which excite only the spectral region of interest (5-9). While the water resonance can be considerably reduced in intensity with the saturation and selective excitation methods, they have the disadvantage that resonances near that of water cannot be observed. Nuclei whose resonances are in this region can be observed with the inversion-recovery method if their spin-lattice relaxation times are different from that of the water protons. We report here a new and effective method with which the water resonance can be completely eliminated and resonances near the water resonance can be observed. In the water attenuation by T2 relaxation (WATR) experiment described here, the spinspin relaxation time of the water protons is selectively reduced by chemical exchange, and the 'H NMR spectrum is measured by the Car-r-Pmcell-Meiboom-Gill (CPMG) pulse sequence (90"(x)-(T-180"(y)-r),-acquisition) (IO). By making the time between the 90" pulse and acquisition of the free induction decay long enough, the water resonance is completely eliminated. Phase modulation of homonuclear coupled multiplet patterns is suppressed by using a sufficiently short time between successive 180" pulses. The development of the WATR experiment follows from our finding that the water resonance can be selectively eliminated from 'H NMR spectra of aqueous suspensions of red blood cells and from aqueous protein solutions with the spin-echo pulse sequence (I I). Several classes of compounds which have labile protons can be used as reagents to selectively decrease the T2 of the water protons by chemical exchange; we describe here the use of NH&l. The rate of exchange of protons between Hz0 and NHd+ (12)