Water Diffusion and Transport in Oil Paints as Studied by Unilateral NMR and 1H High-Resolution MAS-NMR Spectroscopy

Heavy metal carboxylate degradation severely affects thousands of oil paintings. Relative humidity has been reported to accelerate the rate of the reactions. To evaluate its role further, water diffusion and molecular mobility of protons in linseed oil-based lead white paints were studied by unilateral NMR and ¹H HRMAS spectroscopy. The results indicate that exposure to high %RH for relatively long times affects the dynamics of the oil paint's mobile fraction and that the effect is more pronounced as the thickness of the film increases. It was found that the paint can absorb appreciable amounts of water and has a porosity of approximately 6 % available for the diffusion of water, for which a regime of restricted diffusion was observed. Furthermore, the presence of bound and free-moving water, due to the possible formation of hydrated ionic-group clusters, supports the hypothesis of a polymeric/ionomeric network, as well as regions of essentially water free to move as in the bulk. The findings allow a better understanding of the role of water as a factor activating the degradation process in linseed oil-based lead white paints.     

Light-Induced Uncaging for Time-Resolved Observations of Biochemical Reactions by MAS NMR Spectroscopy

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time ³¹P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.     

Molecular Recognition of Rosmarinic Acid from Salvia sclareoides Extracts by Acetylcholinesterase: A New Binding Site Detected by NMR Spectroscopy

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer’s disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC‐MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4′‐O‐glucoside, luteolin 3′,7‐di‐O‐glucoside and luteolin 7‐O‐(6′′‐O‐acetylglucoside) were identified by MS² and MS³ data acquired during the LC‐MSⁿ runs. Quantification of rosmarinic acid by HPLC with diode‐array detection (DAD) showed that the butanol extract is the richest one in this component (134 μg mg^?1 extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE‐bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD‐based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.     

Probing Cell-Surface Interactions in Fungal Cell Walls by High-Resolution 1H-Detected Solid-State NMR Spectroscopy

Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field ¹H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.     

Posttranslational Modifications of Intact Proteins Detected by NMR Spectroscopy: Application to Glycosylation

Posttranslational modifications (PTMs) are an integral part of the majority of proteins. The characterization of structure and function of PTMs can be very challenging especially for glycans. Existing methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications, the inability to identify linkage types and thus chemical structure. We present a direct, robust, and simple NMR spectroscopy method for the detection and identification of PTMs in proteins. No isotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approach is well suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.     

Assessment of a Large Enzyme–Drug Complex by Proton‐Detected Solid‐State NMR Spectroscopy without Deuteration

Solid‐state NMR spectroscopy has recently enabled structural biology with small amounts of non‐deuterated proteins, largely alleviating the classical sample production demands. Still, despite the benefits for sample preparation, successful and comprehensive characterization of complex spin systems in the few cases of higher‐molecular‐weight proteins has thus far relied on traditional ¹³C‐detected methodology or sample deuteration. Herein we show for a 29 kDa carbonic anhydrase:acetazolamide complex that different aspects of solid‐state NMR assessment of a complex spin system can be successfully accessed using a non‐deuterated, 500 μg sample in combination with adequate spectroscopic tools. The shown access to protein structure, protein dynamics, as well as biochemical parameters in amino acid sidechains, such as histidine protonation states, will be transferable to proteins that are not expressible in E. coli.     

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in 1H‐Detected Solid‐State NMR Spectroscopy

¹H‐detection can greatly improve spectral sensitivity in biological solid‐state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of ¹H‐detection by the loss of aliphatic side‐chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for ¹H‐detected ssNMR, and it gives high quality spectra for both side‐chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent ¹H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.     

SEAL by NMR: Glyco‐Based Selenium‐Labeled Affinity Ligands Detected by NMR Spectroscopy

We report a method for the screening of interactions between proteins and selenium‐labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR‐active handle and reports on binding through ⁷⁷Se NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to ¹³C NMR, while the NMR spectral width is ten times larger, yielding little overlap in ⁷⁷Se NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium‐based bioactive compounds, such as glycomimetic drug candidates.     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

Strategies for 1H-Detected Dynamic Nuclear Polarization Magic-Angle Spinning NMR Spectroscopy

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected ¹⁴N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.     

Shift Reagents in NMR Spectroscopy

The number of possible applications of NMR spectroscopy has rapidly increased during the past few years. New fields of applications have been opened by the development of supraconducting solenoids and various spin-decoupling techniques and by the method of “pulsed Fourier transform NMR-spectroscopy”. These methods originate mainly from progress in instrumentation. Recently, another “technique” has been introduced into NMR spectroscopy, which—principally on the basis of chemical and spectroscopic experience—is much less expensive but nevertheless useful. The basic principles, background, and most important applications of this method, known as the “NMR-shift-reagent technique”, form the subject of this paper.     

De Novo 3D Structure Determination from Sub‐milligram Protein Samples by Solid‐State 100 kHz MAS NMR Spectroscopy

Solid‐state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane‐associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10–20 mg each. Here we show that a new NMR probe, pushing magic‐angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well‐defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in‐cell or cell‐free expression.     

Structure-Directing Roles of Organic Molecules in the Formation of Aluminosilicate and Aluminophosphate Molecular Sieves Revealed by 2D 1H DQ-SQ NMR Spectroscopy

Understanding of crystallization mechanisms of molecular sieves is driven by the broad range of usefulness and unique properties they possess. It is still difficult to obtain information related to the crystallization mechanism of molecular sieves, partly because the materials are generally prepared under hydrothermal conditions and the whole reaction happens in the “black box” autoclave. In this work, 2D ¹H DQ-SQ NMR results clearly demonstrate that it is not only the electrostatic interactions between organic structure-directing agents (OSDAs) and the framework, but also the correlation among OSDAs playing the dominant structural directing roles during the crystallization process. Our fundamental understanding of the crystallization mechanism of molecular sieves could be of great value to design and synthesize new molecular sieves with desirable structural properties.     

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.     

High-throughput screening of argan oil composition and authenticity using benchtop 1H NMR

We use 60-MHz benchtop nuclear magnetic resonance (NMR) to acquire ¹H spectra from argan oils of assured origin. We show that the low-field NMR spectrum of neat oil contains sufficient information to make estimates of compositional parameters and to inform on the presence of minor compounds. A screening method for quality and authenticity is presented based on nearest-neighbour outlier detection. A variety of oil types are used to challenge the method. In a survey of retail-purchased oils, several instances of fraud were found.