1D NMR WaterLOGSY as an efficient method for fragment-based lead discovery

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein

Direct observation of hyperpolarization breaking through the spin diffusion barrier

Dynamic nuclear polarization (DNP) is a widely used tool for overcoming the low intrinsic sensitivity of nuclear magnetic resonance spectroscopy and imaging. Its practical applicability is typically bounded, however, by the so-called 'spin diffusion barrier', which relates to the poor efficiency of polarization transfer from highly polarized nuclei close to paramagnetic centers to bulk nuclei. A quantitative assessment of this barrier has been hindered so far by the lack of general methods for studying nuclear-polarization flow in the vicinity of paramagnetic centers. Here we fill this gap and introduce a general set of experiments based on microwave gating that are readily implemented. We demonstrate the versatility of our approach in experiments conducted between 1.2 – 4.2 K in static mode and at 100 K under magic angle spinning (MAS) — conditions typical for dissolution-DNP and MAS-DNP — and for the first time directly observe the dramatic dependence of polarization flow on temperature.The data are organized in subfolders. A PDF document in the root folder summarizes the list of all experiments in the dataset with precisions on experimental parameters and remarks (README.pdf). For each subfolder, the figures of the paper which were produced using the data is the subfolder is listed. Funding provided by: European Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000781Award Number: 714519: HP4allFunding provided by: H2020 Marie Skłodowska-Curie ActionsCrossref Funder Registry ID: http://dx.doi.org/10.13039/100010665Award Number: 766402: ZULFFunding provided by: NSF/DMR and the State of Florida*Crossref Funder Registry ID: Award Number: 1644779Funding provided by: NSF/DMR NIH*Crossref Funder Registry ID: Award Number: S10 OD018519Funding provided by: National Institutes of HealthCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000002Award Number: P41 GM122698 01Funding provided by: National Science FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000001Award Number: CHE 1229170Funding provided by: NSF/DMR and the State of FloridaCrossref Funder Registry ID: Award Number: 1644779Funding provided by: NSF/DMR NIHCrossref Funder Registry ID: Award Number: S10 OD018519All data consist of NMR spectra. Data were collected using high field NMR instruments by Bruker using the software Topspin 3.5.7 and Topspin 3.6.2. They were exported to CSV files

Pulse sequence and sample formulation optimization for dipolar order mediated 1H-13C cross-polarization

We have recently demonstrated the use of contactless radiofrequency pulse sequences under dissolution-dynamic nuclear polarization conditions as an attractive way of transferring polarization from sensitive 1H spins to insensitive 13C spins with low peak radiofrequency pulse powers and energies via a reservoir of dipolar order. However, many factors remain to be investigated and optimized to enable the full potential of this polarization transfer process. We demonstrate herein the optimization of several key factors by: (i) implementing more efficient shaped radiofrequency pulses; (ii) adapting 13C spin labelling; and (iii) avoiding methyl group relaxation sinks. Experimental demonstrations are presented for the case of [1-13C]sodium acetate and other relevant molecular candidates. By employing the range of approaches set out above, polarization transfer using the dipolar order mediated cross-polarization radiofrequency pulse sequence is improved by factors approaching ∼1.65 compared with previous results. Dipolar order mediated 1H→13C polarization transfer efficiencies reaching ∼76% were achieved using significantly reduced peak radiofrequency pulse powers relative to the performance of highly sophisticated state-of-the-art cross-polarization methods, indicating 13C nuclear spin polarization levels on the order of ∼32.1% after 10 minutes of 1H DNP. The approach does not require extensive pulse sequence optimization procedures and can easily accommodate high concentrations of 13C-labelled molecules

An automated system for fast transfer and injection of hyperpolarized solutions

Dissolution dynamic nuclear polarization (dDNP) has become a hyperpolarization method of choice for enhancing nuclear magnetic resonance (NMR) signals. Nuclear spins are polarized in solid frozen samples (in a so-called polarizer) that are subsequently dissolved and transferred to an NMR spectrometer for high sensitivity detection. One of the critical challenges of dDNP is that it requires both a fast transfer to limit nuclear spin relaxation losses as well as stability to guarantee high resolution (no bubbles nor turbulences). Here we describe the design, construction and performances of such a transfer and injection system, that features a 5 m/s speed and sub-Hz spectral resolution upon arrival at the detection spot. We demonstrate the use of such a system for inter-magnet distances of up to 10 m

Approche moléculaire de l'astringence par l'étude des interactions entre les tanins du vin et les protéines de la salve

Lors de la dégustation des vins rouges, une sensation de sécheresse nommée astringence peut avoirlieu. Cette sensation est causée par l’interaction des tanins du vin et des protéines salivairescorrespondant à un phénomène d’une extrême complexité. Cinq tanins représentatifs des vins B1, B2,B3, B4 et C2, et trois peptides représentatifs des protéines de la salive IB714, IB937 et l’histatine 3 ontété synthétisés et étudiés par des approches de physico-chimie et de biologie structurale telles que laRMN, le dichroïsme circulaire et la modélisation moléculaire. Après une étude structurale, lesparamètres de l’interaction ont été déterminés pour l’ensemble des systèmes permettant de construiredes échelles d'affinités montrant l’influence de la structure tridimensionnelle des tanins et de leurnature (degrés de polymérisation), de la longueur du peptide et la meilleure affinité des tanins pour lesPRP comparées aux PRH. Ces études ont également mis en évidence l’importance de la concentrationen tanin sur le phénomène de précipitation. En dessous de leur CMC, les tanins forment des colloïdesavec les peptides par le biais de liaisons hydrogènes, et au-dessus de leur CMC, les interactionshydrophiles restent préférentielles mais des interactions hydrophobes interviennent dans un deuxièmetemps.During tasting of red wines, a sensation of dryness named astringency may occur. This sensation iscaused by the interaction between wine tannins and salivary proteins corresponding to an extremelycomplex phenomenon. Five representative wine tannins B1, B2, B3, B4 and C2, and threerepresentative peptides IB714, IB937 and histatin 3 from saliva were synthesized and studied byphysical chemistry and biology structural tools, such as NMR, circular dichroism and molecularmodeling. After a structural study, the parameters of the interaction were determined for all systemsallowing to build affinities scales, showing the influence of three-dimensional structure of tannins andtheir nature (degree of polymerization), the influence of the peptide length and the higher affinity oftannins for PRP than HRP. These studies have also highlighted the importance of concentration oftannin on the phenomenon of precipitation. Below their CMC, tannins bind specifically to salivaryproteins. Above the CMC, the specific interactions are still present, but tannins can also form micellesand create hydrophobic interactions

Approche moléculaire de l'astringence par l'étude des interactions entre les tanins du vin et les protéines de la salve

Lors de la dégustation des vins rouges, une sensation de sécheresse nommée astringence peut avoirlieu. Cette sensation est causée par l’interaction des tanins du vin et des protéines salivairescorrespondant à un phénomène d’une extrême complexité. Cinq tanins représentatifs des vins B1, B2,B3, B4 et C2, et trois peptides représentatifs des protéines de la salive IB714, IB937 et l’histatine 3 ontété synthétisés et étudiés par des approches de physico-chimie et de biologie structurale telles que laRMN, le dichroïsme circulaire et la modélisation moléculaire. Après une étude structurale, lesparamètres de l’interaction ont été déterminés pour l’ensemble des systèmes permettant de construiredes échelles d'affinités montrant l’influence de la structure tridimensionnelle des tanins et de leurnature (degrés de polymérisation), de la longueur du peptide et la meilleure affinité des tanins pour lesPRP comparées aux PRH. Ces études ont également mis en évidence l’importance de la concentrationen tanin sur le phénomène de précipitation. En dessous de leur CMC, les tanins forment des colloïdesavec les peptides par le biais de liaisons hydrogènes, et au-dessus de leur CMC, les interactionshydrophiles restent préférentielles mais des interactions hydrophobes interviennent dans un deuxièmetemps.During tasting of red wines, a sensation of dryness named astringency may occur. This sensation iscaused by the interaction between wine tannins and salivary proteins corresponding to an extremelycomplex phenomenon. Five representative wine tannins B1, B2, B3, B4 and C2, and threerepresentative peptides IB714, IB937 and histatin 3 from saliva were synthesized and studied byphysical chemistry and biology structural tools, such as NMR, circular dichroism and molecularmodeling. After a structural study, the parameters of the interaction were determined for all systemsallowing to build affinities scales, showing the influence of three-dimensional structure of tannins andtheir nature (degree of polymerization), the influence of the peptide length and the higher affinity oftannins for PRP than HRP. These studies have also highlighted the importance of concentration oftannin on the phenomenon of precipitation. Below their CMC, tannins bind specifically to salivaryproteins. Above the CMC, the specific interactions are still present, but tannins can also form micellesand create hydrophobic interactions

NMR for Fragment-Based Drug Design

International audienc

Overview of probing protein-ligand interactions using NMR

International audienceNuclear magnetic resonance (NMR) is a powerful technique for the study and characterization of protein-ligand interactions. In this unit we review both experiments where the NMR spectrum of the protein is observed (protein-observed NMR experiments) and those where the NMR spectra of the ligand is observed (ligand-observed NMR experiments) for the identification of binding partners, the measurement of protein-ligand affinity, the design of molecules that are active against biological targets such as proteins, and the assessment of the binding modes of the ligands. Ligand-observed methods discussed in this unit are Nuclear Overhauser Effect (NOE)-based approaches, with well-known experiments such as the Saturation Transfer Difference, Water-Ligand Observed via Gradient Spectroscopy (WaterLOGSY), and transferred-Nuclear Overhauser Effect Spectroscopy (tr-NOESY) experiments, and also the INPHARMA experiment. Regarding protein-observed experiments, this unit focuses on the use of chemical shift perturbations observed in protein-NMR spectra upon ligand binding. Also discussed is how these chemical shift perturbations can be used for the analysis of protein-ligand complexes, including fast structure determination when combined with docking