Detection of helical water flows in sub-nanometer channels.

Nanoscale flows of liquids can be revealed in various biological processes and underlie a wide range of nanofluidic applications. Though the integral characteristics of these systems, such as permeability and effective diffusion coefficient, can be measured in experiments, the behaviour of the flows within nanochannels is still a matter of speculation. Herein, we used a combination of quadrupolar solid-state NMR spectroscopy, computer simulation, and dynamic vapour sorption measurements to analyse water diffusion inside peptide nanochannels. We detected a helical water flow coexisting with a conventional axial flow that are independent of each other, immiscible, and associated with diffusion coefficients that may differ up to 3 orders of magnitude. The trajectory of the helical flow is dictated by the screw-like distribution of ionic groups within the channel walls, while its flux is governed by external water vapour pressure. Similar flows may occur in other types of nanochannels containing helicoidally distributed ionic groups and be exploited in various nanofluidic lab-on-a-chip devices.

Valorization of Crab Shells as Potential Sorbent Materials for CO2 Capture.

This study delves into the potential advantage of utilizing crab shells as sustainable solid adsorbents for CO2 capture, offering an environmentally friendly alternative to conventional porous adsorbents, such as zeolites, silicas, metal-organic frameworks (MOFs), and porous carbons. The investigation focuses on crab shell waste, which exhibits inherent natural porosity and N-bearing groups, making them promising candidates for CO2 physisorption and chemisorption applications. Selective deproteinization and demineralization treatments were used to enhance textural properties while preserving the natural porous structure of the crab shells. The impact of deproteinization and demineralization treatments on CO2 adsorption and speciation at the atomic scale, via solid-state NMR, and correlated findings with textural properties and biomass composition were investigated. The best-performing sample exhibits a surface area of 36 m2/g and a CO2 adsorption capacity of 0.31 mmol/g at 1 bar and 298 K, representing gains of ∼3.5 and 2, respectively, compared to the pristine crab shell. These results underline the potential of fishing industry wastes as a cost-effective, renewable, and eco-friendly source to produce functional porous adsorbents.

Unravelling the structure of CO2 in silica adsorbents: an NMR and computational perspective.

This comprehensive review describes recent advancements in the use of solid-state NMR-assisted methods and computational modeling strategies to unravel gas adsorption mechanisms and CO2 speciation in porous CO2-adsorbent silica materials at the atomic scale. This work provides new perspectives for the innovative modifications of these materials rendering them more amenable to the use of advanced NMR methods.

One-Shot Resin 3D-Printed Stators for Low-Cost Fabrication of Magic-Angle Spinning NMR Probeheads.

Additive manufacturing such as three-dimensional (3D)-printing has revolutionized the fast and low-cost fabrication of otherwise expensive NMR parts. High-resolution solid-state NMR spectroscopy demands rotating the sample at a specific angle (54.74°) inside a pneumatic turbine, which must be designed to achieve stable and high spinning speeds without mechanical friction. Moreover, instability of the sample rotation often leads to crashes, resulting in costly repairs. Producing these intricate parts requires traditional machining, which is time-consuming, costly, and relies on specialized labor. Herein, we show that 3D-printing can be used to fabricate the sample holder housing (stator) in one shot, while the radiofrequency (RF) solenoid was constructed using conventional materials available in electronics stores. The 3D-printed stator, equipped with a homemade RF coil, showed remarkable spinning stability, yielding high-quality NMR data. At a cost below 5 €, the 3D-printed stator represents a cost reduction of over 99% compared to repaired commercial stators, illustrating the potential of 3D-printing for mass-producing affordable magic-angle spinning stators.

Contribution of non-ionic interactions on bile salt sequestration by chitooligosaccharides: Potential hypocholesterolemic activity.

Chitooligosaccharides have been suggested as cholesterol reducing ingredients mostly due to their ability to sequestrate bile salts. The nature of the chitooligosaccharides-bile salts binding is usually linked with the ionic interaction. However, at physiological intestinal pH range (6.4 to 7.4) and considering chitooligosaccharides pKa, they should be mostly uncharged. This highlights that other type of interaction might be of relevance. In this work, aqueous solutions of chitooligosaccharides with an average degree of polymerization of 10 and 90 % deacetylated, were characterized regarding their effect on bile salt sequestration and cholesterol accessibility. Chitooligosaccharides were shown to bind bile salts to a similar extent as the cationic resin colestipol, both decreasing cholesterol accessibility as measured by NMR at pH 7.4. A decrease in the ionic strength leads to an increase in the binding capacity of chitooligosaccharides, in agreement with the involvement of ionic interactions. However, when the pH is decreased to 6.4, the increase in charge of chitooligosaccharides is not followed by a significant increase in bile salt sequestration. This corroborates the involvement of non-ionic interactions, which was further supported by NMR chemical shift analysis and by the negative electrophoretic mobility attained for the bile salt-chitooligosaccharide aggregates at high bile salt concentrations. These results highlight that chitooligosaccharides non-ionic character is a relevant structural feature to aid in the development of hypocholesterolemic ingredients.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study.

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Assessing CO2 Capture in Porous Sorbents via Solid-State NMR-Assisted Adsorption Techniques.

Adsorption isotherms obtained through volumetric measurements are widely used to estimate the gas adsorption performance of porous materials. Nonetheless, there is always ambiguity regarding the contributions of chemi- and physisorption processes to the overall retained gas volume. In this work, we propose, for the first time, the use of solid-state NMR (ssNMR) to generate isotherms of CO2 adsorbed onto an amine-modified silica sorbent. This method enables the separation of six individual isotherms for chemi- and physisorbed CO2 components, a feat only possible using the discrimination power of NMR spectroscopy. The adsorption mechanism for each adsorbed species was ascertained by tracking their adsorption profiles at various pressures. The proposed method was validated against conventional volumetric adsorption measurements. The isotherm curves obtained by the proposed ssNMR-assisted approach enable advanced analysis of the sorbents, revealing the potential of variable-pressure NMR experiments in gas adsorption applications.

Exploring Molecular Dynamics of Adsorbed CO2 Species in Amine-Modified Porous Silica by Solid-State NMR Relaxation.

Previous studies on CO2 adsorbents have mainly addressed the identification and quantification of adsorbed CO2 species in amine-modified porous materials. Investigation of molecular motion of CO2 species in confinement has not been explored in depth yet. This work entails a comprehensive study of molecular dynamics of the different CO2 species chemi- and physisorbed at amine-modified silica materials through the determination of the rotating frame spin-lattice relaxation times (T 1ρ) by solid-state NMR. Rotational correlation times (τC) were also estimated using spin relaxation models based on the Bloch, Wangsness, and Redfield and the Bloembergen-Purcell-Pound theories. As expected, the τC values for the two physisorbed CO2 species are considerably shorter (32 and 20 µs) than for the three identified chemisorbed CO2 species (162, 62, and 123 µs). The differences in molecular dynamics between the different chemisorbed species correlate well with the structures previously proposed. In the case of the physisorbed CO2 species, the τC values of the CO2 species displaying faster molecular dynamics falls in the range of viscous liquids, whereas the species presenting slower dynamics exhibit T 1ρ and τC values compatible with a CO2 layer of weakly interacting molecules with the silica surface. The values for chemical shift anisotropy (CSA) and 1H-13C heteronuclear dipolar couplings have also been estimated from T 1ρ measurements, for each adsorbed CO2 species. The CSA tensor parameters obtained from fitting the relaxation data agree with the experimentally measured CSA values, thus showing that the theories are well suited to study CO2 dynamics in silica surfaces.

"Hidden" CO2 in Amine-Modified Porous Silicas Enables Full Quantitative NMR Identification of Physi- and Chemisorbed CO2 Species.

Although spectroscopic investigation of surface chemisorbed CO2 species has been the focus of most studies, identifying different domains of weakly interacting (physisorbed) CO2 molecules in confined spaces is less trivial as they are often indistinguishable resorting to (isotropic) NMR chemical shift or vibrational band analyses. Herein, we undertake for the first time a thorough solid-state NMR analysis of CO2 species physisorbed prior to and after amine-functionalization of silica surfaces; combining 13C NMR chemical shift anisotropy (CSA) and longitudinal relaxation times (T 1). These methods were used to quantitatively distinguish otherwise overlapping physisorbed CO2 signals, which contributed to an empirical model of CO2 speciation for the physi- and chemisorbed fractions. The quantitatively measured T 1 values confirm the presence of CO2 molecular dynamics on the microsecond, millisecond, and second time scales, strongly supporting the existence of up to three physisorbed CO2 species with proportions of about 15%, 15%, and 70%, respectively. Our approach takes advantage from using adsorbed 13C-labeled CO2 as probe molecules and quantitative cross-polarization magic-angle spinning to study both physi- and chemisorbed CO2 species, showing that 45% of chemisorbed CO2 versus 55% of physisorbed CO2 is formed from the overall confined CO2 in amine-modified hybrid silicas. A total of six distinct CO2 environments were identified from which three physisorbed CO2 were discriminated, coined here as "gas, liquid, and solid-like" CO2 species. The complex nature of physisorbed CO2 in the presence and absence of chemisorbed CO2 species is revealed, shedding light on what fractions of weakly interacting CO2 are affected upon pore functionalization. This work extends the current knowledge on CO2 sorption mechanisms providing new clues toward CO2 sorbent optimization.

Targeted DNP for biomolecular solid-state NMR.

High-field dynamic nuclear polarization is revolutionizing the scope of solid-state NMR with new applications in surface chemistry, materials science and structural biology. In this perspective article, we focus on a specific DNP approach, called targeted DNP, in which the paramagnets introduced to polarize are not uniformly distributed in the sample but site-specifically located on the biomolecular system. After reviewing the various targeting strategies reported to date, including a bio-orthogonal chemistry-based approach, we discuss the potential of targeted DNP to improve the overall NMR sensitivity while avoiding the use of glass-forming DNP matrix. This is especially relevant to the study of diluted biomolecular systems such as, for instance, membrane proteins within their lipidic environment. We also discuss routes towards extracting structural information from paramagnetic relaxation enhancement (PRE) induced by targeted DNP at cryogenic temperature, and the possibility to recover site-specific information in the vicinity of the paramagnetic moieties using high-resolution selective DNP spectra. Finally, we review the potential of targeted DNP for in-cell NMR studies and how it can be used to extract a given protein NMR signal from a complex cellular background.

Detecting acetylated aminoacids in blood serum using hyperpolarized 13C-1Η-2D-NMR.

Dynamic Nuclear Polarization (DNP) can substantially enhance the sensitivity of NMR experiments. Among the implementations of DNP, ex-situ dissolution DNP (dDNP) achieves high signal enhancement levels owing to a combination of a large temperature factor between 1.4 and 300 K with the actual DNP effect in the solid state at 1.4 K. For sufficiently long T1 relaxation times much of the polarization can be preserved during dissolution with hot solvent, thus enabling fast experiments during the life time of the polarization. Unfortunately, for many metabolites found in biological samples such as blood, relaxation times are too short to achieve a significant enhancement. We have therefore introduced 13C-carbonyl labeled acetyl groups as probes into amino acid metabolites using a simple reaction protocol. The advantage of such tags is a sufficiently long T1 relaxation time, the possibility to enhance signal intensity by introducing 13C, and the possibility to identify tagged metabolites in NMR spectra. We demonstrate feasibility for mixtures of amino acids and for blood serum. In two-dimensional dDNP-enhanced HMQC experiments of these samples acquired in 8 s we can identify acetylated amino acids and other metabolites based on small differences in chemical shifts.

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites.

Locating binding sites in biomolecular assemblies and solving their structures are of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selective highlighting and identification of residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding is performed using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivities. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidths between 0.5 and 1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g. 13C,15N-labeled protein).

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family.

We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/ J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon-carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.

Novel PTM-TEMPO biradical for fast dissolution dynamic nuclear polarization.

The synthesis and characterization of a novel trityl-TEMPO biradical and the investigation of its properties as Dynamic Nuclear Polarization (DNP) polarizing agent are reported. Comparison with a structurally related monoradical (PTM-TEMPE) or mixtures of the two monoradical components reveals that the biradical has a much higher polarization efficiency and a faster polarization buildup. This offers the possibility of faster recycling further contributing to its efficiency as a polarizing agent.

Application of ex situ dynamic nuclear polarization in studying small molecules.

Dynamic nuclear polarization (DNP) has become an attractive technique to boost the sensitivity of NMR experiments. In the case of ex situ polarizations two-dimensional (2D) spectra are limited by the short lifetime of the polarization after dissolution and sample transfer to a high field NMR magnet. This limitation can be overcome by various approaches. Here we show how the use of (13)C-labelled acetyl tags can help to obtain 2D-HMQC spectra for many small molecules, owing to a nuclear Overhauser enhancement between (13)C spins originating from the long-lived carbonyl carbon, which extends the lifetimes of other (13)C spins with shorter longitudinal relaxation times. We also show an alternative approach of using an optimized polarization matrix.

Quantum rotor induced hyperpolarization.

Despite its broad applicability NMR has always been limited by its inherently low sensitivity. Hyperpolarization methods have the potential to overcome this limitation and, in the case of ex situ dynamic nuclear polarization (DNP), large enhancement factors have been achieved. Although many other polarization methods have been described in the past, including chemically and parahydrogen-induced polarization and optical pumping, DNP has recently been the most popular. Here we present an additional polarization mechanism arising from quantum rotor effects in methyl groups, which generates polarizations at temperatures < 1.5 K and interferes with DNP at such temperatures. The polarization generated by this mechanism is efficiently transferred via carbon bound protons. Although quantum rotor polarizations have been studied for a small range of molecules in great detail, we observe such effects for a much broader range of substances with very different polarization rates at temperatures < 1.5 K. Moreover, we report transfer of quantum rotor polarization across a chain of protons. The observed effect not only influences the polarization in low-temperature DNP experiments but also opens a new independent avenue to generate enhanced sensitivity for NMR.

Optimizing the polarization matrix for ex situ dynamic nuclear polarization.

Although recent advances in dynamic nuclear polarization techniques have boosted the otherwise low sensitivity of NMR spectroscopy, the efficiency of the hyperpolarization process depends on the composition of the polarization matrix, in particular on the contact between the radical and the target molecule and the capability of the matrix to transfer polarization through spin diffusion. A concept for optimal matrix design is presented, applied to obtain two-dimensional heterocorrelated spectra of small drug-like molecules in 1-2 min after 90 min of hyperpolarization.

Light penetration and photoisomerization in rhodopsin studied by numerical simulations and double-quantum solid-state NMR spectroscopy.

The penetration of light into optically thick samples containing the G-protein-coupled receptor rhodopsin is studied by numerical finite-element simulations and double-quantum solid-state NMR experiments. Illumination with white light leads to the generation of the active bathorhodopsin photostate in the outer layer of the sample but generates a large amount of the side product, isorhodopsin, in the sample interior. The overall yield of bathorhodopsin is improved by using monochromatic 420 nm illumination and by mixing the sample with transparent glass beads. The implications of these findings on the interpretation of previously published rhodopsin NMR data are discussed.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision.

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.