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Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process.

Solid-state NMR has become the method of choice for the assessment of protein structure for insoluble objects lacking long-range order. In this context, it is apparent that solid-state NMR is also perfectly poised toward the characterization of immobilized proteins. For these systems, it is possible to understand at the atomic level which perturbations, if any, are occurring as a result of the functionalization. Here we describe how it is possible to accomplish the NMR characterization of enzymes that have been immobilized through different approaches, and we introduce the reader to the choice of the experimental strategy that can be useful in different cases. An outlook on the level of information that can be attained is also given, in view of recent methodological advancements.

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS. Second, the fraction of resonances in the spectrum of a paramagnetic sample that can be paired with the diamagnetic counterparts is critical for the assignment. With the data from two tag positions a reliable assignment could be obtained for 60% of the methyl groups and for many of the remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.

Solid-state NMR is becoming a powerful tool to detect atomic-level structural features of biomolecules even when they are bound to (or trapped in) solid systems that lack long-range three-dimensional order. We here demonstrate that it is possible to probe protein-ligand interactions from a protein-based perspective also when the protein is entrapped in silica, thus translating into biomolecular solid-state NMR all of the considerations that are usually made to understand the chemical nature of the interaction of a protein with its ligands. This work provides a proof of concept that also immobilized enzymes can be used for protein-based NMR protein-ligand interactions for drug discovery.

We present the first solid-state NMR study of an iron-sulfur protein. The combined use of very fast (60 kHz) magic-angle spinning and tailored radiofrequency irradiation schemes allows the detection and the assignment of most of the 1 H and 13 C resonances of the oxidized high-potential iron-sulfur protein I from Ectothiorhodospira halophila (EhHiPIP I), including those in residues coordinating the Fe 4 S 4 cluster. For these residues, contact shifts as large as 100 and 400 ppm for 1 H and 13 C resonances, respectively, were observed, which represent the most shifted solid-state NMR signals ever measured in metalloproteins. Interestingly, by targeting EhHiPIP I in a crystalline environment, we were able to capture distinct paramagnetic signatures from the two conformations present in the asymmetric unit. The magnetic properties of the system were verified by following the temperature dependence of the contact-shifted cysteine resonances.

Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50 kHz), allows for the acquisition of traditional (1)H-(15)N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that (1)H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.

In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution NMR, and even outperform them in the case of large systems. However, several factors affect the spectral quality in SSNMR samples, and not all systems turn out to be equally amenable for this methodology. In this review we attempt at analyzing the causes of this variable behavior and at providing hints to increase the chances of experimental success., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This "tuned" flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

Great theoretical and methodological advances are pushing the limits of resolution and sensitivity in solid state NMR (SSNMR). However, sample preparation remains a critical issue for the success of an experiment. The factors affecting spectral quality in SSNMR samples are discussed, examining cases encountered in the literature and presenting new experimental data. A discussion on resolution and sensitivity in sedimented solutes is framed in this context.

In this review, the use of general NMR spectroscopy techniques to detect ligand binding and to monitor enzyme kinetics and inhibition, which appear particularly useful in hit identification and validation, is reiterated. Furthermore, the use of NMR-based strategies for lead optimisations that are based on either iterative derivatisations of an initial core structure or on linking fragments that occupy adjacent pockets in the target's binding site will also be described. Several recent examples will be reported and the use of these techniques in cases when the three dimensional structure of the target protein is known will be discussed.