SWET for Secure Water Suppression on Probes with High Quality Factor

Water suppression by selective preirradiation is increasingly difficult to achieve on probeheads with high quality factor because of the opposing forces of radiation damping. Here we show that a simple modification to the WET scheme provides reliable water suppression in aqueous solutions of proteins and peptides with minimal saturation of the H^α protons. The scheme is shown to work also with dilute peptide solutions. It is recommended to maintain the water suppression during the evolution time of COSY experiments by weak selective irradiation that causes only minimal Bloch-Siegert shifts. The new water-suppression scheme suppresses the water magnetization by spatial scrambling. Traditional water suppression by preirradiation is similarly based more on water scrambling due to the radiofrequency inhomogeneity than on relaxation effects.Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Improving solvent suppression in jump-return NOESY experiments

Summary The problems associated with solvent suppression in jump-return NOESY spectra and in particular the difficulties experienced with using short mixing times are examined. It is shown that the degree of water suppression depends critically on the extent of radiation damping of the water magnetisation during the mixing time of the NOESY sequence. A new jump-return NOESY sequence is proposed which incorporates field gradients and which achieves good levels of water suppression for all values of the mixing time, and for all increments of the NOESY experiment.     

Selective excitation of intense solvent signals in the presence of radiation damping

Summary Selective water excitation schemes are provided which rely on the radiation damping effect in probeheads characterized by high quality factors. The schemes are implemented in homonuclear NOE and ROE experiments, designed for the selective observation of water-protein cross peaks and their assignment using standard probeheads. The one-dimensional NOE and ROE experiments selectively record the cross section through the water signal usually measured in two-dimensional NOESY and ROESY spectra, and the two-dimensional NOE-NOESY and ROE-NOESY experiments selectively measure the cross section through the water line from 3D NOESY-NOESY and ROESY-NOESY spectra, respectively.     

Biomolecular ligands screening using radiation damping difference WaterLOGSY spectroscopy

Water-ligand observed via gradient spectroscopy (WaterLOGSY) is a widely used nuclear magnetic resonance method for ligand screening. The crucial procedure for the effectiveness of WaterLOGSY is selective excitation of the water resonance. The selective excitation is conventionally achieved by using long selective pulse, which causes partial saturation of the water magnetization leading to reduction of sensitivity, in addition to time consuming and error prone. Therefore, many improvements have been made to enhance the sensitivity and robustness of the method. Here we propose an alternative selective excitation scheme for WaterLOGSY by utilizing radiation damping effect. The pulse scheme starts simply with a hard inversion pulse, instead of selective pulse or pulse train, followed by a pulse field gradient to control the radiation damping effect. The rest parts of the pulse scheme are similar to conventional WaterLOGSY. When the gradient pulse is applied immediately after the inversion pulse, the radiation damping effect is suppressed, and all of the magnetization is inversed. When the gradient pulse and the inversion pulse are about 10–20 ms apart, the radiation damping effect remains active and drives the water magnetization toward +z-axis, resulting in selective non-inversion of the water magnetization. By taking the differences of the spectra obtained under these two conditions, one should get the result of WaterLOGSY. The method is demonstrated to be simple, robust and sensitive for ligand screening.     

Enhanced sensitivity of rapidly exchanging amide protons by improved phase cycling and the constructive use of radiation damping

Summary Two alternative, general methods are presented that lead to enhanced signal intensity of rapidly exchanging protons. Both methods work by avoiding saturation of the water resonance, and are convenient to implement since they do not use any selective pulses. One method carefully chooses proton pulse phases and gradient strength and position in such a way that the water is realigned along the +z axis at the beginning of the acquisition time. An alternative method is proposed for cases where the pulse sequence does not allow such phase cycling. The latter uses radiation damping to bring water back to the +z axis 20–30 ms after acquisition. The methods are applied to the triple-resonance experiments HNCA, HNCO and HN(CO)CA. Both methods require pulsed B₀ field gradients and can result in higher signal intensity by a factor of two or more.     

Slight mistuning of a cryogenic probe significantly perturbs the water <Superscript>1</Superscript>H precession frequency

A shift of the water proton precession frequency is described that can introduce errors in chemical shifts derived using the water signal as the chemical shift reference. This shift, f_s, arises as a consequence of radiation damping when the water proton and detector circuit resonance frequencies differ. Herein it is shown that experimental values of f_s, measured as a function of detector circuit tuning offset for 500 and 900 MHz cryogenic probes, are in good agreement with theory. Of importance is the fact that even a small degree of mistuning, which does not significantly impact the performance of a pulse sequence, introduces chemical shift errors of ±0.03 ppm, that negatively impact many types of experiments. A simple remedy that attenuates the frequency shift is presented.     

Farseer-NMR: automatic treatment,analysis and plotting of large,multi-variable NMR data

Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr–Purcell–Meiboom–Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics. Using a fast folding protein, gpW, we have shown that previously inaccessible kinetics can be accessed with the improved precision and efficiency of the measurement by using this experiment.     

A study of protein-water exchange through the off-resonance ROESY experiment: Application to the DNA-binding domain of AlcR

Summary In this communication a new NMR experiment for the safe observation and quantification of water-protein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shift-based separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.     

The potent pro‐oxidant activity of rhododendrol–eumelanin is enhanced by ultraviolet A radiation

RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some consumers. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products and RD–eumelanin exerts a potent pro‐oxidant activity. Cellular antioxidants were oxidized by RD–eumelanin with a concomitant production of H₂O₂. In this study, we examined whether this pro‐oxidant activity of RD–eumelanin is enhanced by ultraviolet A (UVA) radiation because most RD–induced leukoderma lesions are found in sun‐exposed areas. Exposure to a physiological level of UVA (3.5 mW/cm²) induced a two to fourfold increase in the rates of oxidation of GSH, cysteine, ascorbic acid, and NADH. This oxidation was oxygen‐dependent and was accompanied by the production of H₂O₂. These results suggest that RD–eumelanin is cytotoxic to melanocytes through its potent pro‐oxidant activity that is enhanced by UVA radiation.     

Molecular Cloning and Characterization of 9 cDNAs for Genes That Are Responsive to Desiccation in Arabidopsis thaliana: SequenceAnalysis of One cDNA Clone That Encodes a Putative Transmembrane Channel Protein

We isolated nine cDNAs for genes of Arabidopsis thaliana thatare induced by the effects of water deficit by using the techniqueof differential hybridization and named them RD (RD for Responsiveto Desiccation). The timing of induction of their mRNAs variesbetween these RD genes. Abscisic acid induces mRNAs that correspondto clones designated RD22 and RD29 but not to those designatedRD19, RD21 and RD28, indicating that there are several signal-transductionpathways from water stress to expression of RD genes. Sequenceanalysis of the cDNA clone RD28 revealed that RD28 encodes amembrane protein with sequence homology to the majorintrinsicprotein of bovine lens fiber gap junction, soybean nodulin-26and the glycerolfacilitator from Escherichia coli, indicatingthat the putative RD28 protein is a member of the family oftransmembrane channel proteins. (Received October 31, 1991; Accepted January 13, 1992)     

Tyrosinase‐catalyzed oxidation of rhododendrol produces 2‐methylchromane‐6,7‐dione,the putative ultimate toxic metabolite: implications for melanocyte toxicity

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a skin‐whitening agent until it was reported to induce leukoderma in July 2013. To explore the mechanism underlying its melanocyte toxicity, we characterized the tyrosinase‐catalyzed oxidation of RD using spectrophotometry and HPLC. Oxidation of RD with mushroom tyrosinase rapidly produced RD‐quinone, which was quickly converted to 2‐methylchromane‐6,7‐dione (RD‐cyclic quinone) and RD‐hydroxy‐p‐quinone through cyclization and addition of water molecule, respectively. RD‐quinone and RD‐cyclic quinone were identified as RD‐catechol and RD‐cyclic catechol after NaBH₄ reduction. Autoxidation of RD‐cyclic catechol produced superoxide radical. RD‐quinone and RD‐cyclic quinone quantitatively bound to thiols such as cysteine and GSH. These results suggest that the melanocyte toxicity of RD is caused by its tyrosinase‐catalyzed oxidation through production of RD‐cyclic quinone which depletes cytosolic GSH and then binds to essential cellular proteins through their sulfhydryl groups. The production of ROS through autoxidation of RD‐cyclic catechol may augment the toxicity.     

GUARDD: user-friendly MATLAB software for rigorous analysis of CPMG RD NMR data

Molecular dynamics are essential for life, and nuclear magnetic resonance (NMR) spectroscopy has been used extensively to characterize these phenomena since the 1950s. For the past 15 years, the Carr-Purcell Meiboom-Gill relaxation dispersion (CPMG RD) NMR experiment has afforded advanced NMR labs access to kinetic, thermodynamic, and structural details of protein and RNA dynamics in the crucial μs-ms time window. However, analysis of RD data is challenging because datasets are often large and require many non-linear fitting parameters, thereby confounding assessment of accuracy. Moreover, novice CPMG experimentalists face an additional barrier because current software options lack an intuitive user interface and extensive documentation. Hence, we present the open-source software package GUARDD (Graphical User-friendly Analysis of Relaxation Dispersion Data), which is designed to organize, automate, and enhance the analytical procedures which operate on CPMG RD data (). This MATLAB-based program includes a graphical user interface, permits global fitting to multi-field, multi-temperature, multi-coherence data, and implements χ ²-mapping procedures, via grid-search and Monte Carlo methods, to enhance and assess fitting accuracy. The presentation features allow users to seamlessly traverse the large amount of results, and the RD Simulator feature can help design future experiments as well as serve as a teaching tool for those unfamiliar with RD phenomena. Based on these innovative features, we expect that GUARDD will fill a well-defined gap in service of the RD NMR community.     

Radiation damping compensation of selective pulses in water–protein exchange spectroscopy

The observation of nuclear Overhauser effects (NOEs) between bound water and biological macromolecules such as proteins and nucleic acids can be improved by inverting the water resonance selectively while compensating for radiation damping effects. The efficiency of inversion, the offset profiles, and the appearance of 2D NOE-NOESY spectra can be improved in comparison with earlier methods.     

RD20, a stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana

Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.     

Strong Suppression of Surface Plasmon Radiation Damping Rate in Noble Metal Nanoshells

Radiation damping of surface plasmon oscillations in metallic nanoparticles is proportional to their volume. For relatively large particles, this canal dominates the other mechanisms of relaxation and becomes the main limiting factor for spectral sensitivity of nanoparticles. In this communication, we consider metallic nanoshell with the dielectric core and calculate the radiation damping rate of surface plasmon oscillations, depending on the geometry and dielectric constants of the surrounding environment and the core. It is shown that surface plasmon radiation damping in nanoshell is suppressed by several orders of magnitude as compared to the solid particle of the same outer radius. This effect is conditioned by strong redshift of surface plasmon frequencies with the decrease of shell thickness. It is also demonstrated that the radiation damping rate of core–shell particle is highly sensitive with respect to the refractive index of surrounding media.     

Gradient-enhanced TOCSY experiments with improved sensitivity and solvent suppression

Summary Gradient-enhanced versions of the homonuclear TOCSY experiment are described, with solvent suppression and sensitivity superior to that of a conventional TOCSY experiment. The pulse sequences are constructed by appending a WATERGATE module to a z-filtered TOCSY experiment. Pulsed-field gradients and appropriately phased selective rf pulses are used to maintain precise control of the water magnetization vector. Problems associated with radiation damping and spin-locking of the water magnetization are thus alleviated. The water magnetization is returned to equilibrium prior to each acquisition, which improves water suppression and minimizes signal losses due to saturation transfer.     

Water-protein NOEs: Optimized scheme for selective water excitation

An alternative scheme for selective water excitation is proposed. The pulse sequence saturates the resonances from the solute, allowing the observation of water-solute NOEs with low artifact levels. The water resonance is subsequently excited by a relatively non-selective 90° pulse. The scheme is compared to other selective water excitation schemes. 2D NOE-NOESY and ROE-NOESY pulse sequences are proposed which afford high sensitivity by efficient water excitation and flip-back by radiation damping, yet allow the use of short mixing times for the buildup of water-solute NOEs.     

Suppression of radiation damping during selective excitation of the water signal: The WANTED sequence

Summary A new pulse sequence is presented allowing the use of long selective pulses at the water frequency using standard equipment. Radiation damping is suppressed during the pulse by the use of gradient echoes programmed between the single pulses of a DANTE train. This WANTED (water-selective DANTE using gradients) sequence thus allows the observation of interactions with water without the use of special probe heads or filtering of undesired resonances. By combining the WANTED sequence with NOESY, ROESY and NOESY-GSQC experiments, we obtain selective 1D and 2D spectra fit to the observation of chemical exchange and dipolar interactions between water and protein protons.     

Neutralization of radiation damping by selective feedback on a 400 MHz NMR spectrometer

Summary Radiation damping is a phenomenon well known among NMR spectroscopists of proteins as a source of undesirable features, especially in high-field and high-Q probe NMR. In this paper, we present an electronic neutralization network which dramatically reduces radiation damping. It detects the radiation field profile and feeds back into the probe an rf field with identical amplitude and opposite phase. Experimental results of a practical implementation carried out on a 400 MHz Bruker spectrometer are shown.