Solution-state NMR Studies Research Articles

Mapping the binding site of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1.

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is expressed in erythrocytes, vascular endothelial cells, and immune cells. KCa3.1 is involved in a wide variety of disease pathologies, suggesting that both positive and negative gating modulators would be of interest as pharmacological tools and potential drugs. NS309 is the most potent positive gating modulator for KCa3.1, with an EC50 of 75 nM, and interestingly, it is capable of increasing channel activity even at saturating calcium concentrations. However, its binding site is currently unknown. Previous X-ray crystallographic and solution-state NMR studies suggested that NS309 was binding between calmodulin's N-lobe and the C-terminal region of the calmodulin binding domain and interacting with residues M51 and R372. Unfortunately, the crystal dimer used in these studies was later found to be an artifact and disproved when the full-length crystal structure of KCa3.1 was published in 2018. This full-length structure suggested that the “real” binding site for positive gating modulators was located within the interface between calmodulin's N-lobe and the S4-S5 linker, a hypothesis we have now confirmed via site-directed mutagenesis and whole-cell patch clamp. In addition, we have also been investigating a second binding pocket between the distal end of the S4-S5 intracellular loop and the S6 transmembrane segment (a site highlighted by Schroedinger's SiteMap software). And while NS309 does not seem to be binding in this second pocket, several mutations within this pocket cause the channel to become hypersensitive to calcium, indicating that this pocket could serve as another functional binding site for positive gating channel modulators.

Site-selective post-modification of short α/γ hybrid foldamers: a powerful approach for molecular diversification towards biomedical applications.

The extensive research work in the exhilarating area of foldamers (artificial oligomers possessing well-defined conformation in solution) has shown them to be promising candidates in biomedical research and materials science. The post-modification approach is successful in peptides, proteins, and polymers to modulate their functions. To the best of our knowledge, site-selective post-modification of a foldamer affording molecules with different pendant functional groups within a molecular scaffold has not yet been reported. We demonstrate for the first time that late-stage site-selective functionalization of short hybrid oligomers is an efficient approach to afford molecules with diverse functional groups. In this article, we report the design and synthesis of hybrid peptides with repeating units of leucine (Leu) and 5-amino salicylic acid (ASA), regioselective post-modification, conformational analyses (based on solution-state NMR, circular dichroism and computational studies) and morphological studies of the peptide nanostructures. As a proof-of-concept, we demonstrate the applications of differently modified peptides as drug delivery agents, imaging probes, and anticancer agents. The novel feature of the work is that the difference in reactivity of two phenolic OH groups in short biomimetic peptides was utilized to achieve site-selective post-modification. It is challenging to apply the same approach to short α-peptides having a poor folding tendency, and their post-functionalization may considerably affect their conformation.

Reactivity of Waterlogged Archeological Elm Wood with Organosilicon Compounds Applied as Wood Consolidants: 2D 1H-13C Solution-State NMR Studies.

Some organosilicon compounds, including alkoxysilanes and siloxanes, proved effective in stabilizing the dimensions of waterlogged archaeological wood during drying, which is essential in the conservation process of ancient artifacts. However, it was difficult to determine a strong correlation between the wood stabilizing effect and the properties of organosilicon compounds, such as molecular weight and size, weight percent gain, and the presence of other potentially reactive groups. Therefore, to better understand the mechanism behind the stabilization effectiveness, the reactivity of organosilicons with wood polymers was studied using a 2D 1H–13C solution-state NMR technique. The results showed an extensive modification of lignin through its demethoxylation and decarbonylation and also the absence of the native cellulose anomeric peak in siloxane-treated wood. The most substantial reactivity between wood polymers and organosilicon was observed with the (3-mercaptopropyl)trimethoxysilane treatment, showing complete removal of lignin side chains, the lowest syringyl/guaiacyl ratio, depolymerization of cellulose and xylan, and reactivity with the C6 primary hydroxyls in cellulose. This may explain the outstanding stabilizing effectiveness of this silane and supports the conclusion that extensive chemical interactions are essential in this process. It also indicates the vital role of a mercapto group in wood stabilization by organosilicons. This 2D NMR technique sheds new light on the chemical mechanisms involved in organosilicon consolidation of wood and reveals what chemical characteristics are essential in developing future conservation treatments.

Sample volume effects in optical overhauser dynamic nuclear polarization

The optical dynamic nuclear polarization (DNP) method has been proposed as an alternative to microwave pumping as a hyperpolarization method for solution-state NMR studies. Using continuous laser illumination to photogenerate triplet states in the presence of a persistent radical produces chemically-induced dynamic electron polarization (CIDEP) via the radical-triplet pair mechanism (RTPM), with cross-relaxation transferring this to nuclear hyperpolarization via an Overhauser mechanism. Numerical simulations have previously indicated that reducing the sample volume while maintaining a constant optical density can significantly increase the NMR signal enhancement, due to the larger steady-state concentration of triplets obtained. Here we provide the first experimental confirmation of these effects, producing a nearly five-fold increase in the optical DNP enhancement factor just by reducing the sample volume with optimal dye and radical concentrations adjusted for each optical path length. The results are supported with an in depth analysis of volume effects in the numerical model, with which they are in good qualitative agreement. These important observations will impact on the future development of the technique, with particular significance for attempts to apply DNP methods to increase sensitivity for volume-limited biological samples.

A Non-Canonical Linker between Zinc Fingers 10 and 11 of Miz-1 Promotes Conformational Exchange: Implications for the Mechanism of Recognition of the Consensus DNA Sequence

Miz-1 (ZBTB17) is a poly-Zinc Finger BTB/POZ transcription factor with 12 consecutive C2H2 Zinc Fingers (ZFs). Miz-1 binds transcriptional start sites (TSS), through a consensus sequence, to regulate the expression of genes involved in the cell cycle, autophagy, trafficking as well as T- and B-cell development. Although it is not known how exactly Miz-1 recognises this consensus, evidence and predictions suggest that ZFs 7 to 12 are able to bind it contiguously. As observed in the other members of the ZBTB family, striking divergences in highly conserved positions in the primary structures of the ZFs and linkers of Miz-1 are present. In fact, compared to the conserved TGEKP/Y found in the rest of ZFs 7-12, the linker between ZFs 10 and 11 has the following sequence: DNIRP/H586. We show by solution-state NMR and 15N-relaxation studies that this linker generates two coupled conformational exchange phenomena on the “biological” μs-ms timescale. On the one hand, we demonstrate that the His residue (H586) weakens the βα tertiary structure in ZF11 and promotes the population of an opened state in which the inter-ZF contacts, necessary for canonical DNA binding, is lacking. The existence of this opened state is supported by the local unfolding of the first helical turn of the recognition helix of ZF11. On the other hand, although this local unfolding is quenched by the replacement of the His by the conserved Tyr residue (Y), we found that another conformational exchange is present in the DNIRP linker. Most interestingly, we observe that the DNIRP linker adopts a helical structure that also impedes interZF contacts necessary for canonical and contiguous binding of the consensus. This implies that the recognition of the consensus by ZFs 7-12 is accomplished in a segmented fashion. Supported by bioinformatics predictions and DNA binding assays, our structural and dynamical data are congruent with a model whereby ZFs 7-9 and 11-12 can independently recognize both ends and strands of the consensus. Finally, considering the dynamic nature of DNA and the conformational exchange in ZFs 10-11, we further demonstrate that the simultaneous binding of ZF 7-9 or ZFs 11-12 to their respective subsites is physically possible in a coiled conformation of the consensus.

Evaluation of Sn(II) aminoalkoxide precursors for atomic layer deposition of SnO thin films.

We have successfully prepared and structurally characterized a family of eight tin(II) heteroleptic complexes, [Sn(NR2)(ON)]x (NR2 = NMe2 (1a-d) or N(SiMe3)2 (2a-d); x = 1 or 2) and four homoleptic systems, [Sn(κ2-ON)2] (3a-d) from a series of aminoalcohols and fluorinated aminoalcohols (H{ON}) having a different number of methyl/trifluoromethyl substituents at the α-carbon atom, [HOC(R1)(R2)CH2NMe2] (R1 = R2 = H (H{dmae}) (a); R1 = H, R2 = Me (H{dmap}) (b); R1 = R2 = Me (H{dmamp}) (c); R1 = R2 = CF3 (H{Fdmamp}) (d)). The synthetic route used reactions of either [Sn{N(SiMe3)2}2] or [Sn(NMe2)2] with one or two equivalents of the aminoalcohols (a-d) in dry aprotic solvents leading to elimination of amines and formation of the Sn(II) species 1a-d, 2a-d and 3a-d respectively. All complexes were thoroughly characterized by NMR spectroscopy (1H, 13C, 19F, and 119Sn) as well as single-crystal X-ray diffraction studies. In all case the solid state molecular structures of the complexes have been unambiguously established: the solid state structures 1a-b and 1c are dimeric with central {Sn2N2} cores resulting from bridging {μ2-NMe2} units, in which the Sn(II) atoms are four-coordinate. In contrast, the solid state structures of complexes 1c and 2a-c possess similarly dimeric structures, with four-coordinate Sn(II) atoms, in which the oxygen atoms of the {ON} ligand bridge two Sn(II) centres to form dimers with a central {Sn2O2} core. Uniquely in this study, 2d, [Sn(κ2-O,N-OCMe2CH2NMe2){N(SiMe3)2}] is monomeric with a three coordinate Sn(II) centre. The homoleptic complexes 3a-d are all isostructural with monomeric four-coordinate structures with disphenoidal geometries. Solution state NMR studies reveal complicated ligand exchange processes in the case of the heteroleptic complexes 1a-d and 2a-d. Contrastingly, the homoleptic systems 3a-d show no such behaviour. While complexes 1a-d and 2a-d displayed either poor thermal stability or multistep thermal decomposition processes, the thermal behaviour of the homoleptic complexes, 3a-d, was investigated in order to determine the effects, if any, of the degree of fluorination and asymmetry of the aminoalkoxide ligands on the suitability of these complexes as ALD precursors for the deposition of SnO thin films.

Solution-state NMR study of organic components of industrial Ziegler-Natta catalysts: Effect of by-products on catalyst performance

Chemical routes for preparing Ziegler-Natta (ZN) catalysts accompany the formation of by-products that remain in final catalysts in different amounts. In the history of ZN catalysts, much less emphasis has been given to analyze such by-products present in catalysts. Herein, we prepared various Mg(OEt)2-based ZN catalysts and conducted solution-state NMR measurements to characterize and quantify the organic components of these catalysts. It was found that, depending on the molecular structure of the internal donor (ID) and catalyst preparation conditions, an ID can undergo side reactions to produce organic by-products. All the catalysts contained different amounts of titanium alkoxy by-products formed in the reaction between Mg(OEt)2 and TiCl4, which were detrimental for the catalyst activity. These by-products were responsible for increasing the amount of the amorphous fraction in polypropylene and decreasing the highly crystalline fraction's stereoregularity. Possible hypotheses to explain the negative impacts of titanium alkoxy by-products are discussed.

Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD-box RNA helicase DbpA from E. coli

ATP-dependent DEAD-box helicases constitute one of the largest families of RNA helicases and are important regulators of most RNA-dependent cellular processes. The functional core of these enzymes consists of two RecA-like domains. Changes in the interdomain orientation of these domains upon ATP and RNA binding result in the unwinding of double-stranded RNA. The DEAD-box helicase DbpA from E. coli is involved in ribosome maturation. It possesses a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA-like domains. The RRM recruits DbpA to nascent ribosomes by binding to hairpin 92 of the 23S rRNA. To follow the conformational changes of Dbpa during the catalytic cycle we initiated solution state NMR studies. We use a divide and conquer approach to obtain an almost complete resonance assignment of the isoleucine, leucine, valine, methionine and alanine methyl group signals of full length DbpA (49 kDa). In addition, we also report the backbone resonance assignments of two fragments of DbpA that were used in the course of the methyl group assignment. These assignments are the first step towards a better understanding of the molecular mechanism behind the ATP-dependent RNA unwinding process catalyzed by DEAD-box helicases.

Sugar-derived oxazolone pseudotetrapeptide as γ-turn inducer and anion-selective transporter.

The intramolecular cyclization of a C-3-tetrasubstituted furanoid sugar amino acid-derived linear tetrapeptide afforded an oxazolone pseudo-peptide with the formation of an oxazole ring at the C-terminus. A conformational study of the oxazolone pseudo-peptide showed intramolecular C=O···HN(II) hydrogen bonding in a seven-membered ring leading to a γ-turn conformation. This fact was supported by a solution-state NMR and molecular modeling studies. The oxazolone pseudotetrapeptide was found to be a better Cl−-selective transporter for which an anion–anion antiport mechanism was established.

Unravelling the Role of S100A9 in the Development of Neurodegenerative Disease

S100A9 is a pro-inflammatory calcium binding protein released by neutrophils in response to neuroinflammatory events. These events can be triggered by a range of conditions including traumatic brain injury and bacterial infection but are also observed in a range of neurodegenerative conditions including Alzheimer's Disease and Parkinson's Disease. S100A9 is intrinsically amyloidogenic self-aggregating to form organised fibrils both in vitro and in vivo. These S100A9 fibrils congregate with amyloid-β to seed and accelerate the growth of amyloid-β fibrils, as observed in in vitro studies. S100A9 possesses two Ca2+ binding sites which when vacant increase its propensity to aggregate into an ordered fibrillar state. Inversely, increasing calcium concentration inhibits S100A9 fibril formation as verified by Thioflavin-T assay. Our solution-state NMR studies indicate that in the absence of calcium the protein is largely unfolded adopts a number of interconverting states which become more structured upon calcium binding. Interestingly complementary synchrotron radiation circular dichroism measurements suggest a high alpha-helical content irrespective of calcium levels, suggesting that these interconverting species are rich in short-lived helical states. To determine the structural transition that occur upon fibril formation, solid-state NMR investigations have been utilised. These studies demonstrate that S100A9 fibrils formed in vitro have a single well-ordered structure with mobile regions limited to 15-25 residues in size. Analysis of the chemical shifts indicate the protein exists as a single polymorph with a rigid protein core that is predominantly β-strand in structure. Employing paramagnetic relaxation assisted data collection (PAC) progress is being made towards a complete assignment permitting a detailed analysis of the conformation of S100A9 within amyloid fibres.

Facile synthesis of stable selenocystine peptides and their solution state NMR studies.

A facile general route for the synthesis of various selenocystine tripeptides containing acidic, basic and neutral side chain amino acids is reported. Here, TFA labile side chain protected selenocysteine has been used as a precursor for the synthesis of selenopeptides. The peptides are highly stable in dimethyl sulphoxide, thus enabling detailed NMR studies by solution phase 1- and 2-dimensional NMR spectroscopy.

Assigning methyl resonances for protein solution-state NMR studies.

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.

Atom Efficient Magnesiation of N‐Substituted Alkyl Indoles with a Mixed Sodium‐Magnesium Base

This study presents the alkali metal mediated magnesiation (AMMMg) of three N‐alkylated indoles with the mixed Na/Mg base [(TMEDA)Na(TMP)2Mg(CH2SiMe3)] 1 (TMEDA = N,N,N′,N′‐tetramethylethylenediamine, TMP = 2,2,6,6‐tetramethylpiperidine). All three magnesiated indoles have been successfully characterised by single‐crystal X‐ray diffraction and solution state NMR studies, whereas iodolysis and Pd‐catalysed cross coupling have been investigated. The steric nature of the N‐alkyl group changes the reactivity and efficiency of 1 to give either atom efficient disodium tetraindol‐2‐ylmagnesiates [(Na‐TMEDA)2Mg(α‐C9H8N)4] 2 and [(Na‐TMEDA)2Mg(α‐C10H11N)4] 3, or [(TMEDA)Na(TMP)(α‐C11H12N)Mg(TMP)] 4, whereby only one indole molecule is selectively deprotonated.

Slow spin crossover in bis-meridional Fe2+ complexes through spin-state auto-adaptive N6/N8 coordination.

Fe2+ spin crossover (SCO) complexes with long-lived excited high-spin (HS) states are promising molecular switches. An enhanced kinetic stability of spin-state isomers can be expected to foster applications beyond the limits of cooperative SCO. In this study, we describe a new approach to slow down the spin-state exchange by simple commutation of a phenyl substituent by a pyridyl substituent. To this end, N4 ligand 6-(3-(pyridin-2-yl)-1H-pyrazol-1-yl)-2,2'-bipyridine (3b) is synthesized as an N4 homologue of the well-established meridional N3 ligands motif. Phenyl-substituted 6-(3-phenyl-1H-pyrazol-1-yl)-2,2'-bipyridine (3a) serves as an intrinsic N3 reference throughout. 3b offers variable coordination numbers, N3 versus N3(+1) and N4, reflecting the preferences of the metal center. As is shown herein through an extended solid-state structure-chemical and solution-state NMR study, which is augmented by density-functional theory modeling, both the coordination geometry and its structural dynamics are indeed highly sensitive towards the expansion of the nominal donor number. The additional donors in 3b introduced through the phenyl-pyridine commutation actually give rise to a rich and diverse stereochemistry of the derived Zn2+ and Fe2+ complexes. Notably, even within a single complex unit coordination of 3b ranges from strongly distorted N3 coordination with a long assisting additional contact (Zn2+ and Fe2+) to a more symmetric N2(+2) or N4 situation in Fe2+. DFT modeling unravels that the additional donors are hemi-labile and coordinate to the Fe2+ only in HS state, leaving the elusive low-spin (LS) state in a fairly undisturbed octahedral environment with 3b being N3 coordinate. That is, the coordination number of the complex autogeneously responds to the altered spin-state. Necessarily this switch in coordination number requires strong structural changes upon SCO. This leads to increased activation barriers for SCO as could be deduced from a temperature-dependent analysis of the dynamic 1H NMR-line broadening and corroborated by accompanying theoretical analysis of the SCO reaction coordinate. For [Fe(3b)2]2+ long spin-state lifetimes τ > 1 ms prevail below the characteristic temperature T (1 ms) = 235 K; this value should be compared with a lifetime of only 150 ns derived for the close analogue [Fe(3a)2]2+. The principle applied herein is general and allows transferring of LS Fe2+ complexes with suitably placed phenyl substituents into SCO complexes with spin-state adaptive coordination number and hence long-lived HS excited states.

Bis-meridional Fe2+ spincrossover complexes of phenyl and pyridyl substituted 2-(pyridin-2-yl)-1,10-phenanthrolines.

A series of Fe2+ SCO complexes of substituted 2-(pyridin-2-yl)-1,10-phenanthrolines 2 was prepared and the SCO (spincrossover) properties were characterized in the solid state (X-ray crystallography, SQUID magnetometry) and in solution (VT-1H NMR spectroscopy), augmented by theoretical modelling. Bis-meridional coordination of the tridendate 2a-c and tetradentate 2d ligands gives octahedral and distorted trigonal-dodecahedral complexes [Fe(2)2]2+, respectively, which were identified as SCO complexes with the transition temperature T1/2 below room temperature. SCO in the solid state is limited to bromo-substituted [Fe(2a)2]2+ (Dalton Trans., 2017, 46, 6218-6229) and [Fe(2b)2]2+ with a pyridine-appended phenyl group, whereas solution state NMR studies reveal SCO behaviour for all complexes, which is in agreement with DFT derived results. As anticipated from its N6(+2) coordination in the HS state, DFT structure modelling of [Fe(2d)2]2+ identified deviation from a structure-conserving SCO reaction coordinate; that is, Fe-N breathing is accompanied by a change in the coordination number. Accordingly, a remarkably slow SCO is observed in [Fe(2d)2]2+, owing to an extended coordinate. De-novo defined characteristic temperatures T(τHSLS) are introduced as structure-dependent parameters deemed to define the onset of phenomenological "slow" SCO. The rich phenomenology of the NMR spectra of [Fe(2)2]2+ is identified to be largely controlled by the dynamics of spin-state exchange and a qualitative illustration of the NMR-reporters of SCO is suggested.

A Tetramer Derived from Islet Amyloid Polypeptide.

Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic β-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.

The Structure of the Diatom Silaffin Peptide R5 within Freestanding Two-Dimensional Biosilica Sheets.

The silaffin peptide R5 is instrumental to the mineralization of silica cell walls of diatom organisms. The peptide is also widely employed in biotechnology, for example, in the encapsulation of enzymes and for fusion proteins in tissue regeneration. Despite its scientific and technological importance, the interfacial structure of R5 during silica precipitation remains poorly understood. We herein elucidate the conformation of the peptide in its active form within silica sheets by interface-specific vibrational spectroscopy in combination with molecular dynamics simulations. Contrary to previous solution-state NMR studies, our data confirm that R5 maintains a defined structure when interacting with extended silica sheets. We show that the entire amino acid sequence of R5 interacts with silica during silica formation, leading to the intercalation of silica into the assembled peptide film.

A ladder coordination polymer based on Ca(2+) and (4,5-dicyano-1,2-phenylene)bis(phosphonic acid): crystal structure and solution-state NMR study.

The preparation of coordination polymers (CPs) based on either transition metal centres or rare-earth cations has grown considerably in recent decades. The different coordination chemistry of these metals allied to the use of a large variety of organic linkers has led to an amazing structural diversity. Most of these compounds are based on carboxylic acids or nitrogen-containing ligands. More recently, a wide range of molecules containing phosphonic acid groups have been reported. For the particular case of Ca(2+)-based CPs, some interesting functional materials have been reported. A novel one-dimensional Ca(2+)-based coordination polymer with a new organic linker, namely poly[[diaqua[μ4-(4,5-dicyano-1,2-phenylene)bis(phosphonato)][μ3-(4,5-dicyano-1,2-phenylene)bis(phosphonato)]dicalcium(II)] tetrahydrate], {[Ca2(C8H4N2O6P2)2(H2O)2]·4H2O}n, has been prepared at ambient temperature. The crystal structure features one-dimensional ladder-like ∞(1)[Ca2(H2cpp)2(H2O)2] polymers [H2cpp is (4,5-dicyano-1,2-phenylene)bis(phosphonate)], which are created by two distinct coordination modes of the anionic H2cpp(2-) cyanophosphonate organic linkers: while one molecule is only bound to Ca(2+) cations via the phosphonate groups, the other establishes an extra single connection via a cyano group. Ladders close pack with water molecules through an extensive network of strong and highly directional O-H...O and O-H...N hydrogen bonds; the observed donor-acceptor distances range from 2.499 (5) to 3.004 (6) Å and the interaction angles were found in the range 135-178°. One water molecule was found to be disordered over three distinct crystallographic positions. A detailed solution-state NMR study of the organic linker is also provided.

Characterization of fast-tumbling isotropic bicelles by PFG diffusion NMR.

Small isotropic bicelles are versatile membrane mimetics, which, in contrast to micelles, provide a lipid bilayer and are at the same time suitable for solution-state NMR studies. The lipid composition of the bilayer is flexible allowing for incorporation of various head groups and acyl chain types. In bicelles, lipids are solubilized by detergents, which are localized in the rim of the disk-shaped lipid bilayer. Bicelles have been characterized by a broad array of biophysical methods, pulsed-field gradient NMR (PFG NMR) being one of them. PFG NMR can readily be used to measure diffusion coefficients of macromolecules. It is thus employed to characterize bicelle size and morphology. Even more importantly, PFG NMR can be used to study the degree of protein association to membranes. Here, we present the advances that have been made in producing small, fast-tumbling isotropic bicelles from a variety of lipids and detergents, together with insights on the morphology of such mixtures gained from PFG NMR. Furthermore, we review approaches to study protein-membrane interaction by PFG NMR. Copyright © 2015 John Wiley & Sons, Ltd.

Urinary excretion and metabolism of miglustat and valproate in patients with Niemann–Pick type C1 disease: One- and two-dimensional solution-state 1H NMR studies

Niemann–Pick type C1 (NP-C1) disease is a neurodegenerative lysosomal storage disease for which the only approved therapy is miglustat (MGS). In this study we explored the applications and value of both one- and two-dimensional high-resolution NMR analysis strategies to the detection and quantification of MGS and its potential metabolites in urine samples collected from NP-C1 disease patients (n=47), and also applied these techniques to the analysis of the anticonvulsant drug valproate and one of its major metabolites in ca. 30% of these samples (i.e. from those who were also receiving this agent for the control of epileptic seizures). A combination of high-resolution 1D and 2D TOCSY/NOESY techniques confirmed the identity of MGS in the urinary 1H NMR profiles of NP-C1 patients treated with this agent (n=25), and its quantification was readily achievable via electronic integration of selected 1D resonance intensities. However, this analysis provided little or no evidence for its metabolism in vivo, observations consistent with those acquired in corresponding experiments performed involving an in vitro microsomal system. Contrastingly, the major valproate metabolite 1-O-valproyl-β-glucuronide was readily detectable and quantifiable in 14/47 of the urine samples investigated, despite some resonance overlap problems (identification of this agent was confirmed by experiments involving equilibration of these samples with β-glucuronidase, a process liberating free valproate). In order to facilitate and validate the detection of MGS in urine specimens, full assignments of the 1H NMR spectra of MGS in both buffered aqueous (pH 7.10) and deuterated methanol solvent systems were also made. The pharmacological and bioanalytical significance of data acquired are discussed, with special reference to the advantages offered by high-resolution NMR analysis.