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Here it is described nanogels (NG) based on a chitosan matrix, which are covalently stabilized by a bisamide derivative of Mn-t-CDTA (t-CDTA = trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid). the Mn(II) complex acts both as a contrast medium and as a cross-linking agent. These nanogels are proposed as an alternative to the less stable paramagnetic nanogels obtained by electrostatic interactions between the polymeric matrix and paramagnetic Gd(III) chelates. The present novel nanogels show: i) relaxivity values seven times higher than that of typical monohydrated Mn(II) chelates at the clinical fields, thanks to the combination of a restricted mobility of the complex with a fast exchange of the metal-bound water molecule; ii) high stability of the formulation over time at pH 5 and under physiological conditions, thus excluding metal leaking or particles aggregation; iii) good extravasation and accumulation, with a maximum contrast achieved at 24 h post-injection in mice bearing subcutaneous breast cancer tumor; iv) high T 1 contrast (1 T) in the tumor 24 h post-injection. These improved properties pave the way for the use of these paramagnetic nanogels as promising magnetic resonance imaging (MRI) probes for in vitro and in vivo preclinical applications., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones., (© 2021 Wiley-VCH GmbH.)

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y] 6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT 2 R/AT 1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NMR spectroscopy is an indispensable technique for the determination of the chemical identity and structure of small molecules. The method is especially recognized for its robustness and intrinsically quantitative nature, and has manifested itself as a key analytical platform for diverse fields of application, ranging from chemical synthesis to metabolomics. Unfortunately, the slow recovery of nuclear spin polarization by spin-lattice (T 1 ) relaxation causes most experimental time to be lost on idle waiting. Furthermore, truly quantitative NMR (qNMR) spectroscopy requires waiting times of 5-times the longest T 1 in the sample, making qNMR spectroscopy slow and inefficient. We demonstrate here that co-solute paramagnetic relaxation can mitigate these two problems simultaneously. The addition of a small amount of paramagnetic gadolinium chelate, available in the form of commercial contrast-agent solutions, enables cheap, quantitative, and efficient high-throughput mixture analysis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metabolomics deals with the whole ensemble of metabolites (the metabolome). As one of the -omic sciences, it relates to biology, physiology, pathology and medicine; but metabolites are chemical entities, small organic molecules or inorganic ions. Therefore, their proper identification and quantitation in complex biological matrices requires a solid chemical ground. With respect to for example, DNA, metabolites are much more prone to oxidation or enzymatic degradation: we can reconstruct large parts of a mammoth's genome from a small specimen, but we are unable to do the same with its metabolome, which was probably largely degraded a few hours after the animal's death. Thus, we need standard operating procedures, good chemical skills in sample preparation for storage and subsequent analysis, accurate analytical procedures, a broad knowledge of chemometrics and advanced statistical tools, and a good knowledge of at least one of the two metabolomic techniques, MS or NMR. All these skills are traditionally cultivated by chemists. Here we focus on metabolomics from the chemical standpoint and restrict ourselves to NMR. From the analytical point of view, NMR has pros and cons but does provide a peculiar holistic perspective that may speak for its future adoption as a population-wide health screening technique., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Scope: According to Eurostat 2016, approximately 119 million European citizens live at-risk-of-poverty (ROP). This subpopulation is highly diverse by ethnicity, age, and culture in the different EU states, but they all have in common a low income that could represent an increased risk of nutrient deficiencies due to poor nutritional habits. This study aims to investigate the human urine metabolome in the search of common biomarkers representing dietary deficiencies amongst European populations at ROP., Methods and Results: 2732 urine samples were collected from 1391 subjects across five different European countries, including the United Kingdom, Finland, Italy, Lithuania, and Serbia, and analyzed using 1 H-NMR spectroscopy. The resulting urine metabolome data were explored according to study design factors including economic status, country, and gender., Conclusion: Partitioning of the effects derived from the study design factors using ANOVA-simultaneous component analysis (ASCA) revealed that country and gender effects were responsible for most of the systematic variation. The effect of economic status was, as expected, much weaker than country and gender, but more pronounced in Lithuania than in other countries. Citrate and hippurate were among the most powerful ROP biomarkers. The possible relationship between these markers and nutritional deficiencies amongst the ROP population is discussed., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO 2 F) with the ϵ-NH 2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH 2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO 2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO 2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO 2 -NH) linkage., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsically disordered proteins (IDPs) experience a diverse spectrum of motions that are difficult to characterize with a single experimental technique. Herein we combine high- and low-field nuclear spin relaxation, nanosecond fluorescence correlation spectroscopy (nsFCS), and long molecular dynamics simulations of alpha-synuclein, an IDP involved in Parkinson disease, to obtain a comprehensive picture of its conformational dynamics. The combined analysis shows that fast motions below 2 ns caused by local dihedral angle fluctuations and conformational sampling within and between Ramachandran substates decorrelate most of the backbone N-H orientational memory. However, slow motions with correlation times of up to ca. 13 ns from segmental dynamics are present throughout the alpha-synuclein chain, in particular in its C-terminal domain, and global chain reconfiguration occurs on a timescale of ca. 60 ns. Our study demonstrates a powerful strategy to determine residue-specific protein dynamics in IDPs at different time and length scales., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbohydrate-based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life-threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T-cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide-protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid-state NMR spectroscopy. As a test case, a large protein assembly, l-asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The design and synthesis of the Ln 3+ complexes of a DOTA-containing (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) inhibitor of matrix metalloproteinases are reported. The tight binding of the sulfonamide scaffold to the catalytic domain of the investigated matrix metalloproteinase is not impaired by the presence of the Ln 3+ -DOTA moiety. The paramagnetic properties of the Ln 3+ complex are exploited to obtain insights into the structural features of the ligand-protein interactions and to evaluate the influence of the linker length on the quality of the paramagnetic restraints., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Long-range pseudo-contact NMR shifts (PCSs) provide important restraints for the structure refinement of proteins when a paramagnetic metal center is present, either naturally or introduced artificially. Here we show that ab initio quantum-chemical methods and a modern version of the Kurland-McGarvey approach for paramagnetic NMR (pNMR) shifts in the presence of zero-field splitting (ZFS) together provide accurate predictions of all PCSs in a metalloprotein (high-spin cobalt-substituted MMP-12 as a test case). Computations of 314 13 C PCSs using g- and ZFS tensors based on multi-reference methods provide a reliable bridge between EPR-parameter- and susceptibility-based pNMR formalisms. Due to the high sensitivity of PCSs to even small structural differences, local structures based either on X-ray diffraction or on various DFT optimizations could be evaluated critically by comparing computed and experimental PCSs. Many DFT functionals provide insufficiently accurate structures. We also found the available 1RMZ PDB X-ray structure to exhibit deficiencies related to binding of a hydroxamate inhibitor. This has led to a newly refined PDB structure for MMP-12 (5LAB) that provides a more accurate coordination arrangement and PCSs., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report our recent efforts directed at improving high-field DNP experiments. We investigated a series of thiourea nitroxide radicals and the associated DNP enhancements ranging from ε = 25 to 82 that demonstrate the impact of molecular structure on performance. We directly polarized low-gamma nuclei including 13 C, 2 H, and 17 O using trityl via the cross effect. We discuss a variety of sample preparation techniques for DNP with emphasis on the benefit of methods that do not use a glass-forming cryoprotecting matrix. Lastly, we describe a corrugated waveguide for use in a 700 MHz / 460 GHz DNP system that improves microwave delivery and increases enhancements up to 50%.

A new strategy to exploit galectin presence to target matrix metalloproteinases (MMPs) is presented. A bifunctional conjugate with lactose and an inhibitor for MMPs is able to bind MMP and Gal-3 simultaneously. This compound might allow the lectin to attract the MMP inhibitor to the tumour site and to block protumoural activities of the lectin at the same time., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Fibers and prebiotics represent a useful dietary approach for modulating the human gut microbiome. Therefore, aim of the present study was to investigate the impact of four flours (wholegrain rye, wholegrain wheat, chickpeas and lentils 50:50, and barley milled grains), characterized by a naturally high content in dietary fibers, on the intestinal microbiota composition and metabolomic output., Methods and Results: A validated three-stage continuous fermentative system simulating the human colon was used to resemble the complexity and diversity of the intestinal microbiota. Fluorescence in situ hybridization was used to evaluate the impact of the flours on the composition of the microbiota, while small-molecule metabolome was assessed by NMR analysis followed by multivariate pattern recognition techniques. HT29 cell-growth curve assay was used to evaluate the modulatory properties of the bacterial metabolites on the growth of intestinal epithelial cells. All the four flours showed positive modulations of the microbiota composition and metabolic activity. Furthermore, none of the flours influenced the growth-modulatory potential of the metabolites toward HT29 cells., Conclusion: Our findings support the utilization of the tested ingredients in the development of a variety of potentially prebiotic food products aimed at improving gastrointestinal health., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The S100 protein family is a highly conserved group of Ca(2+)-binding proteins that belong to the EF-hand type and are considered potential drug targets. In the present study we focused our attention on two members of the family: S100A13 and S100B; the former is involved in the nonclassical protein release of two proangiogenic polypeptides FGF-1 and IL-1alpha that are involved in inflammatory processes, whereas S100B is known to interact with the C-terminal domain of the intracellular tumor suppressor p53 and promote cancer development. We screened, using waterLOGSY NMR experiments, 430 molecules of a generic fragment library and we identified different hits for each protein. The subset of fragments interacting with S100B has very few members in common with the subset interacting with S100A13. From the (15)N-HSQC NMR spectra of the proteins in the presence of those hits the chemical shift differences Deltadelta(HN) were calculated, and the main regions of surface interaction were identified. A relatively large variety of interaction regions for various ligands were identified for the two proteins, including known or suggested protein-protein interaction sites.

Deoxymyoglobin has been investigated by NMR spectroscopy to determine the magnetic anisotropy through pseudocontact shifts and the total magnetic susceptibility through Evans measurements. The magnetic anisotropy values were found to be Deltachi(ax)=-2.03+/-0.08 x 10(-32) m(3) and Deltachi(rh)=-1.02+/-0.09 x 10(-32) m(3). The negative value of the axial susceptibility anisotropy originates from the z tensor axis lying in the heme plane, unlike all other heme systems investigated so far. This magnetic axis is almost exactly orthogonal to the axial histidine plane. The other two axes lie essentially in the histidine plane, the closest to the heme normal being tilted by about 36 degrees from it, towards pyrrole A on the side of the proximal histidine. From the comparison with cytochrome c' it clearly appears that the position of the one axis lying in the heme plane is related to the axial histidine orientation. Irrespective of the directions, the magnetic anisotropy is smaller than that of the analogous reduced cytochrome c' and of the order of that of low-spin iron(III). The magnetic anisotropy of the system permits the measurement of residual dipolar couplings, which, together with pseudocontact shifts, prove that the solution structure is very similar to that in the crystalline state. Magnetic measurements, at variance with previous data, demonstrate that there is an orbital contribution to the magnetic moment, micro(eff)=5.5 micro(B). Finally, from the magnetic anisotropy data, the hyperfine shifts of iron ligands could be separated in pseudocontact and contact components, and hints are provided to understand the spin-delocalisation mechanism in S=2 systems by keeping in mind the delocalisation patterns in low-spin S=1/2 and high-spin S= 5/2 iron(III) systems.