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.     

Amplification of radiation damping in a 600-MHz NMR spectrometer: Application to the study of water-protein interactions

Summary A new application of a recently developed electronic radiation-damping (RD) control system is presented. It is possible to amplify radiation damping so as to make the water magnetization return back to its equilibrium direction in a time shorter than the characteristic RD time. Certain types of experiments involving radiation damping as a selective inversion pulse can be significantly improved by this new method. Moreover, amplification of RD is shown to improve water suppression and consequently the dynamics of 2D NOESY experiments on proteins.     

WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.     

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.     

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water*

A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H₂O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.     

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.     

Alternate HMQC experiments for recording HN and HC-correlation spectra in proteins at high throughput

Alternate implementations of the SOFAST-HMQC experiment are described. In these alternate SOFAST-HMQC experiments (ALSOFAST-HMQC) excitation of the magnetization of interest is achieved by non-selective rf pulses while preserving the equilibrium polarization of passive spins. This alternate excitation scheme also allows the incorporation of a novel sensitivity enhancement protocol which has been most recently developed by Brutscher and coworkers and which permits simultaneous detection of both the x- and y-components of the indirectly detected t(1)-interferograms without the need to introduce additional rf pulses and delays. We show that the ALSOFAST HC-HMQC experiment, which implements an alternate means of frequency selection, enables the detection of methyl resonances with large secondary proton chemical shifts. This is achieved by selecting coherences of interest via a frequency selective carbon inversion pulse. Detailed comparisons between SOFAST- and the presented ALSOFAST-HMQC experiment reveals a considerable degree of mutual complementarity.     

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.     

Measurement of amide hydrogen exchange rates with the use of radiation damping

A simple method for measuring amide hydrogen exchange rates is presented, which is based on the selective inversion of water magnetization with the use of radiation damping. Simulations show that accurate exchange rates can be measured despite the complications of radiation damping and cross relaxation to the exchange process between amide and water protons. This method cannot eliminate the contributions of the exchange-relayed NOE and direct NOE to the measured exchange rates, but minimize the direct NOE contribution. In addition, the amides with a significant amount of such indirect contributions are possible to be identified from the shape of the exchange peak intensity profiles or/and from the apparent relaxation rates of amide protons which are extracted from fitting the intensity profiles to an equation established here for our experiment. The method was tested on ubiquitin and also applied to an acyl carrier protein. The amide exchange rates for the acyl carrier protein at two pHs indicate that the entire protein is highly dynamic on the second timescale. Low protection factors for the residues in the regular secondary structural elements also suggest the presence of invisible unfolded species. The highly dynamic nature of the acyl carrier protein may be crucial for its interactions with its substrate and enzymes.     

Use of a water flip-back pulse in the homonuclear NOESY experiment

Summary A simple modification to the WATERGATE water suppression scheme [Piotto, M., Saudek, V. and Sklená, V. (1992) J. Biomol. NMR, 2, 661–665] is proposed. Radiation damping is used as an active element during the mixing time of a NOESY experiment, in order to obtain a reproducable state of the water magnetization at the end of the mixing time. Through the use of a water flip-back pulse and a gradient-tailored excitation scheme, we obtain both an excellent water suppression and a water magnetization close to equilibrium at the beginning of the acquisition time. We show experimentally that this modification results in a 20% gain in intensity for all signals when using a relaxation delay of 1.5 s, and also that avoiding a semisaturated state for the water magnetization allows the amide protons as well as other proton resonances to relax to equilibrium with their proper relaxation time.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Summary A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H₂O at 5 °C is presented.     

Metabolomic analysis using optimized NMR and statistical methods

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.     

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.     

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.     

Minimisation of sensitivity losses due to the use of gradient pulses in triple-resonance NMR of proteins

Summary The use of pulsed field gradients in multiple-pulse NMR experiments has many advantages, including the possibility of obtaining excellent water suppression without the need for selective presaturation. In such gradient experiments the water magnetization is dephased deliberately; exchange between the saturated protons of the solvent water and the NH protons of a protein transfers this saturation to the protein. As the solvent is in large excess and relaxes relatively slowly, the result is a reduction in the sensitivity of the experiment due to the fact that the NH proton magnetization is only partially recovered. These effects can be avoided by ensuring that the water magnetization remains intact and is returned to the +z-axis at the start of data acquisition. General procedures for achieving this aim in any triple-resonance experiment are outlined and two specific examples are given. Experimental results confirm the sensitivity advantage of the modified sequences.     

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly ¹³C/¹⁵N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s^–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide ¹⁵N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H₂O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin.     

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.     

Ultraviolet resonance Raman study of drug binding in dihydrofolate reductase, gyrase, and catechol O-methyltransferase.

This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as a means of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acids tyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selective enhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change in microenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductase with its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demonstrated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in those systems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/or possible dipole-dipole and dipole-polarizability interactions with the ligand.