2D Solid-state NMR Research Articles

Mediation mechanism of tyrosine 185 on the retinal isomerization equilibrium and the proton release channel in the seven-transmembrane receptor bacteriorhodopsin

Electrostatic coupling leading to conformational changes in proteins is challenging to demonstrate directly, it requires that both the local, discrete electronic details and dynamic information relevant to the functional descriptions are probed. Here, as a novel study to address this challenge, the roles of an aromatic residue in influencing the functional conformational changes of a membrane receptor in its natural membrane environment are reported. Previously intractable discrete electronic details have been obtained using 2D solid-state NMR of specifically labelled receptor, reinforced with molecular dynamic simulations, mutational analysis and functional assays, supported by and compared with rigid-atom crystal structural models. Hydrogen bonding and hydrophobic interactions are identified as the mechanistic origin for direct electromechanical coupling to the dynamics of conformational changes within the receptor.

Microporous Aluminophosphate ULM-6: Synthesis, NMR Assignment, and Its Transformation to AlPO4-14 Molecular Sieve

A pure fluorinated aluminophosphate [Al8P8O32F4·(C3H12N2)2(H2O)2] (ULM-6) has been synthesized via an aminothermal strategy, in which triisopropanolamine (TIPA) is used as the solvent together with the addition of propyleneurea and HF. The 13C NMR spectrum demonstrates that 1,3-diaminopropane, the in situ decomposer of propyleneurea, is the real structure-directing agent (SDA) for ULM-6 crystals. The local Al, P, and F environments of the dehydrated ULM-6 are investigated by 1D and 2D solid-state NMR spectroscopy. The spatial proximities are extracted from 19F{27Al}, 19F{31P}, 27Al{19F}, and 31P{19F} rotational-echo double resonance (REDOR) NMR experiments as well as 19F → 31P heteronuclear correlation (HETCOR) NMR and {31P}27Al HMQC NMR experiments, allowing a full assignment of all the 19F, 27Al, and 31P resonances to the corresponding crystallographic sites. Moreover, it is found that the structure of ULM-6 is closely related to that of AlPO4-14. A combination of high-temperature powder XRD, thermal an...

13C NMR assignments of regenerated cellulose from solid-state 2D NMR spectroscopy

From the assignment of the solid-state 13C NMR signals in the C4 region, distinct types of crystalline cellulose, cellulose at crystalline surfaces, and disordered cellulose can be identified and quantified. For regenerated cellulose, complete 13C assignments of the other carbon regions have not previously been attainable, due to signal overlap. In this study, two-dimensional (2D) NMR correlation methods were used to resolve and assign 13C signals for all carbon atoms in regenerated cellulose. 13C-enriched bacterial nanocellulose was biosynthesized, dissolved, and coagulated as highly crystalline cellulose II. Specifically, four distinct 13C signals were observed corresponding to conformationally different anhydroglucose units: two signals assigned to crystalline moieties and two signals assigned to non-crystalline species. The C1, C4 and C6 regions for cellulose II were fully examined by global spectral deconvolution, which yielded qualitative trends of the relative populations of the different cellulose moieties, as a function of wetting and drying treatments.

Cellulose Structural Polymorphism in Plant Primary Cell Walls Investigated by High-Field 2D Solid-State NMR Spectroscopy and Density Functional Theory Calculations.

The native cellulose of bacterial, algal, and animal origins has been well studied structurally using X-ray and neutron diffraction and solid-state NMR spectroscopy, and is known to consist of varying proportions of two allomorphs, Iα and Iβ, which differ in hydrogen bonding, chain packing, and local conformation. In comparison, cellulose structure in plant primary cell walls is much less understood because plant cellulose has lower crystallinity and extensive interactions with matrix polysaccharides. Here we have combined two-dimensional magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (solid-state NMR) spectroscopy at high magnetic fields with density functional theory (DFT) calculations to obtain detailed information about the structural polymorphism and spatial distributions of plant primary-wall cellulose. 2D (13)C-(13)C correlation spectra of uniformly (13)C-labeled cell walls of several model plants resolved seven sets of cellulose chemical shifts. Among these, five sets (denoted a-e) belong to cellulose in the interior of the microfibril while two sets (f and g) can be assigned to surface cellulose. Importantly, most of the interior cellulose (13)C chemical shifts differ significantly from the (13)C chemical shifts of the Iα and Iβ allomorphs, indicating that plant primary-wall cellulose has different conformations, packing, and hydrogen bonding from celluloses of other organisms. 2D (13)C-(13)C correlation experiments with long mixing times and with water polarization transfer revealed the spatial distributions and matrix-polysaccharide interactions of these cellulose structures. Celluloses f and g are well mixed chains on the microfibril surface, celluloses a and b are interior chains that are in molecular contact with the surface chains, while cellulose c resides in the core of the microfibril, outside spin diffusion contact with the surface. Interestingly, cellulose d, whose chemical shifts differ most significantly from those of bacterial, algal, and animal cellulose, interacts with hemicellulose, is poorly hydrated, and is targeted by the protein expansin during wall loosening. To obtain information about the C6 hydroxymethyl conformation of these plant celluloses, we carried out DFT calculations of (13)C chemical shifts, using the Iα and Iβ crystal structures as templates and varying the C5-C6 torsion angle. Comparison with the experimental chemical shifts suggests that all interior cellulose favor the tg conformation, but cellulose d also has a similar propensity to adopt the gt conformation. These results indicate that cellulose in plant primary cell walls, due to their interactions with matrix polysaccharides, and has polymorphic structures that are not a simple superposition of the Iα and Iβ allomorphs, thus distinguishing them from bacterial and animal celluloses.

Novel insights from NMR spectroscopy into seasonal changes in the composition of dissolved organic matter exported to the Bering Sea by the Yukon River

Seasonal (spring freshet, summer–autumn, and winter) variability in the chemical composition of dissolved organic matter (DOM) from the Yukon River was determined using advanced one- and two-dimensional (2D) solid-state NMR spectroscopy, coupled with isotopic measurements and UV–visible spectroscopy. Analyses were performed on two major DOM fractions, the hydrophobic organic acid (HPOA) and transphilic organic acid (TPIA) fractions obtained using XAD resins. Together these two fractions comprised 64–74% of the total DOM. Carboxyl-rich alicyclic molecules (CRAM) accounted for the majority of carbon atoms in the HPOA (63–77%) and TPIA (54–78%) samples, and more so in winter and summer than in spring samples. 2D and selective NMR data revealed association of abundant nonprotonated O-alkyl and quaternary alkyl C (OCnp, OCnpO and Cq, 13–17% of HPOA and 15–20% of TPIA) and isolated O–CH structures with CRAM, which were not recognized in previous studies. Spectral editing and 2D NMR allowed for the discrimination of carbohydrate-like O-alkyl C from non-carbohydrate O-alkyl C. Whereas two spring freshet TPIA samples contained carbohydrate clusters such as carboxylated carbohydrates (16% and 26%), TPIA samples from other seasons or HPOA samples mostly had small amounts (<8%) of sugar rings dispersed in a nonpolar alkyl environment. Though nonprotonated aromatic C represented the largest fraction of aromatic C in all HPOA/TPIA isolates, only a small fraction (∼5% in HPOA and 3% in TPIA) was possibly associated with dissolved black carbon. Our results imply a relatively stable portion of DOM exported by the Yukon River across different seasons, due to the predominance of CRAM and their associated nonprotonated C–O and O–C–O structures, and elevated reactivity (bio- and photo-lability) of spring DOM due to the presence of terrestrial inputs enriched in carbohydrates and aromatic structures.

Spectral editing at ultra-fast magic-angle-spinning in solid-state NMR: facilitating protein sequential signal assignment by HIGHLIGHT approach.

This study demonstrates a novel spectral editing technique for protein solid-state NMR (SSNMR) to simplify the spectrum drastically and to reduce the ambiguity for protein main-chain signal assignments in fast magic-angle-spinning (MAS) conditions at a wide frequency range of 40-80 kHz. The approach termed HIGHLIGHT (Wang et al., in Chem Comm 51:15055-15058, 2015) combines the reverse (13)C, (15)N-isotope labeling strategy and selective signal quenching using the frequency-selective REDOR pulse sequence under fast MAS. The scheme allows one to selectively observe the signals of "highlighted" labeled amino-acid residues that precede or follow unlabeled residues through selectively quenching (13)CO or (15)N signals for a pair of consecutively labeled residues by recoupling (13)CO-(15)N dipolar couplings. Our numerical simulation results showed that the scheme yielded only ~15% loss of signals for the highlighted residues while quenching as much as ~90% of signals for non-highlighted residues. For lysine-reverse-labeled micro-crystalline GB1 protein, the 2D (15)N/(13)Cα correlation and 2D (13)Cα/(13)CO correlation SSNMR spectra by the HIGHLIGHT approach yielded signals only for six residues following and preceding the unlabeled lysine residues, respectively. The experimental dephasing curves agreed reasonably well with the corresponding simulation results for highlighted and quenched residues at spinning speeds of 40 and 60 kHz. The compatibility of the HIGHLIGHT approach with fast MAS allows for sensitivity enhancement by paramagnetic assisted data collection (PACC) and (1)H detection. We also discuss how the HIGHLIGHT approach facilitates signal assignments using (13)C-detected 3D SSNMR by demonstrating full sequential assignments of lysine-reverse-labeled micro-crystalline GB1 protein (~300 nmol), for which data collection required only 11 h. The HIGHLIGHT approach offers valuable means of signal assignments especially for larger proteins through reducing the number of resonance and clarifying multiple starting points in sequential assignment with enhanced sensitivity.

超1 GHz NMR システムの開発

We have developed the world's highest magnetic-field 1,020 MHz (1.02 GHz) NMR by using a Bi-2223 hightemperature superconducting inner coil and low-temperature superconducting outer coils. High resolution 2D solid-state NMR spectra were achieved for a membrane protein by using the 1.02 GHz NMR. The NMR spectral resolution achieved using the 1.02 GHz NMR was 2.6-fold higher than that achieved using a 700 MHz NMR. This paper describes the historical significance and future prospects of NMR magnets operated beyond 1 GHz.

Solvation and Desolvation Phenomenon and in-Situ NMR Studies on Stripping/Plating of Magnesium Metal in Magnesium Batteries

Multivalent batteries are promising technologies that can potentially outperform lithium ion batteries in term of energy density, power density and unit cost.1 Among which magnesium batteries have attracted much attention over the last a few years. However, scientific challenges continue primarily due to the lack of understanding of the intercalation mechanism of multivalent ions.2 Our recent study is devoted to the understanding of the solvation and desolvation effect with 1H and 2D solid-state NMR spectroscopy by probing the movement of the water molecules and solvent molecules in the system (Bis(trifluoromethan sulfonyl)Imide dissolved in diglyme based magnesium batteries) at various charge states. It is suggested that the water and diglyme molecules play a crucial role in facilitating the intercalation of the magnesium ions in the cathode lattice upon charging and discharging. 3 In addition, the effective stripping and plating effect on the magnesium metal is also investigated. By taking advantage of in situ NMR spectroscopy, we investigate metal vs metal pseudo cells while the cells are polarized. No significant changes in the NMR spectra between the pristine and cycled pseudo cell for Grignard electrolytes, which is in agreements with reversible smooth stripping and plating. 1 D. Aurbach, Z. Lu, A. Schechter, Y. Gofer, H. Gizbar, R. Turgeman, Y. Cohen, M. Moshkovich, E. Levi, Nature 2000, 407, 724-727 10.1038/35037553. 2 H. D. Yoo, I. Shterenberg, Y. Gofer, G. Gershinsky, N. Pour, D. Aurbach, Energy & Environmental Science 2013, 6, 2265-2279 10.1039/c3ee40871j. 3 S. H. Lapidus, N. N. Rajput, X. Qu, K. W. Chapman, K. A. Persson, P. J. Chupas, Physical Chemistry Chemical Physics 2014, 16, 21941-21945 10.1039/c4cp03015j.

Determination of relative orientation between 1H CSA tensors from a 3D solid-state NMR experiment mediated through 1H/1H RFDR mixing under ultrafast MAS

To obtain piercing insights into inter and intramolecular H-bonding, and π-electron interactions measurement of 1H chemical shift anisotropy (CSA) tensors is gradually becoming an obvious choice. While the magnitude of CSA tensors provides unique information about the local electronic environment surrounding the nucleus, the relative orientation between these tensors can offer further insights into the spatial arrangement of interacting nuclei in their respective three-dimensional (3D) space. In this regard, we present a 3D anisotropic/anisotropic/isotropic proton chemical shift (CSA/CSA/CS) correlation experiment mediated through 1H/1H radio frequency-driven recoupling (RFDR) which enhances spin diffusion through recoupled 1H–1H dipolar couplings under ultrafast magic angle spinning (MAS) frequency (70kHz). Relative orientation between two interacting 1H CSA tensors is obtained by fitting two-interacting 1H CSA tensors by fitting two-dimensional (2D) 1H/1H CSA/CSA spectral slices through extensive numerical simulations. To recouple 1H CSAs in the indirect frequency dimensions of a 3D experiment we have employed γ-encoded radio frequency (RF) pulse sequence based on R-symmetry (R1887) with a series of phase-alternated 2700°–90180° composite-180° pulses on citric acid sample. Due to robustness of applied 1H CSA recoupling sequence towards the presence of RF field inhomogeneity, we have successfully achieved an excellent 1H/1H CSA/CSA cross-correlation efficiency between H-bonded sites of citric acid.

Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples.

We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living (15)N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through (15)N-(15)N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish (15)N-(15)N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI-HETCOR and 3D PISEMAI-HETCOR-mixing experiments.

Structural characterization of 13C-enriched humins and alkali-treated 13C humins by 2D solid-state NMR

Complementary (2D) NMR techniques provide new insights into the molecular structure of humin by-products.

Accurate measurements of ¹³C-¹³C distances in uniformly ¹³C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy.

Application of sets of (13)C-(13)C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important (13)C-(13)C distances in uniformly (13)C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ((13)C') and aliphatic ((13)C(aliphatic)) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly (13)C,(15)N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of (13)C'-(13)C(aliphatic) distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform (13)C,(15)N-labeling on the FGAIL fragment.

Silica morphogenesis by lysine-leucine peptides with hydrophobic periodicity.

The use of biomimetic approaches in the production of inorganic nanostructures is of great interest to the scientific and industrial community due to the relatively moderate physical conditions needed. In this vein, taking cues from silaffin proteins used by unicellular diatoms, several studies have identified peptide candidates for the production of silica nanostructures. In the current article, we study intensively one such silica-precipitating peptide, LKα14 (Ac-LKKLLKLLKKLLKL-c), an amphiphilic lysine/leucine repeat peptide that self-organizes into an α-helical secondary structure under appropriate concentration and buffer conditions. The suggested mechanism of precipitation is that the sequestration of hydrophilic lysines on one side of this helix allows interaction with the negatively charged surface of silica nanoparticles, which in turn can aggregate further into larger structures. To investigate the process, we carry out 1D and 2D solid-state NMR (ssNMR) studies on samples with one or two uniformly (13)C- and (15)N-labeled residues to determine the backbone and side-chain chemical shifts. We also further study the dynamics of two leucine residues in the sequence through (13)C spin-lattice relaxation times (T1) to determine the impact of silica coprecipitation on their mobility. Our results confirm the α-helical secondary structure in both the neat and silica-complexed states of the peptide, and the patterns of chemical shift and relaxation time changes between the two states suggest possible mechanisms of self-aggregation and silica precipitation.

BSH-CP based 3D solid-state NMR experiments for protein resonance assignment

We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO-CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO-CA transfer block can be incorporated in a set of three-dimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for resonance assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous resonance assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO-CA transfer schemes in well-established 3D ssNMR approaches for resonance assignment of solid biomolecules.

Structure determination of the theophylline–nicotinamide cocrystal: a combined powder XRD, 1D solid-state NMR, and theoretical calculation study

The crystal structure of the theophylline–nicotinamide cocrystal is determined for the first time by using a combined multi-technique approach.

Demonstration of a common indole-based aromatic core in natural and synthetic eumelanins by solid-state NMR.

Despite the essential functions of melanin pigments in diverse organisms and their roles in inspiring designed nanomaterials for electron transport and drug delivery, the structural frameworks of the natural materials and their biomimetic analogs remain poorly understood. To overcome the investigative challenges posed by these insoluble heterogeneous pigments, we have used l-tyrosine or dopamine enriched with stable (13)C and (15)N isotopes to label eumelanins metabolically in cell-free and Cryptococcus neoformans cell systems and to define their molecular structures and supramolecular architectures. Using high-field two-dimensional solid-state nuclear magnetic resonance (NMR), our study directly evaluates the assumption of structural commonality between synthetic melanin models and the corresponding natural pigments, demonstrating a common indole-based aromatic core in the products from contrasting synthetic protocols for the first time.

Ferrocene in the metal–organic framework MOF-5 studied by homo- and heteronuclear correlation NMR and MD simulation

Advanced solid-state 2D NMR spectroscopy and molecular dynamics computation are employed to investigate the interaction between adsorbed ferrocene molecules and the MOF-5 lattice. Relayed 13C–1H heteronuclear correlation (HETCOR) 2D NMR spectra clearly indicate short-distance contacts between the ferrocene guests and the benzene-1,4-dicarboxylic-acid linkers, mediated via intermolecular 1 H spin diffusion. By use of 2D 1H–1H correlation spectroscopy the distances between 1H nuclei in the guests and the linkers are estimated to be shorter than 0.5nm. MD computer simulations support the interpretation of the 2D solid state NMR studies. Moreover, they suggest a wide distribution of intermolecular distances in this host–guest system with the shortest intermolecular hydrogen–hydrogen distances of 0.15nm.

A Solid-State NMR Study of Amorphous Ezetimibe Dispersions in Mesoporous Silica

The purpose of this work is to examine the ability of methods based on multinuclear and multidimensional solid-state NMR (SSNMR) to perform detailed characterization of amorphous dispersions of ezetimibe adsorbed on mesoporous silica. Ezetimibe was loaded into two types of mesoporous silica with average pore sizes of 2.5 and 21 nm. The mesoporous materials were characterized by powder X-ray diffraction (PXRD), vibrational spectroscopy, differential scanning calorimetry, and (1)H, (13)C, (19)F, and (29)Si SSNMR analysis including relaxation time measurements. Interactions between the drug and silica were investigated using 1D and 2D SSNMR methods based on dipolar correlation using cross-polarization (CP) and spin diffusion. PXRD was used to show the absence of crystalline ezetimibe in the mesoporous materials, and (19)F SSNMR was used to assess drug physical state and study mobility. (19)F-(29)Si CP methods were used to directly detect adsorbed ezetimibe. (1)H-(13)C, (1)H-(19)F, and (1)H-(29)Si, and heteronuclear correlation and (1)H homonuclear correlation experiments were used to investigate interactions between the drug and silica through (1)H environments. SSNMR methods were able to detect interactions between the drug and the silica substrate. Differences between the drug loaded onto silica with two different pore sizes were observed, including differences in hydrogen bonding environment and molecular mobility. These methods should be useful for characterization of similar systems.

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-(13)C, (15)N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during (15)N-(13)C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-(13)C, (15)N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils.

Polymer-derived SiCN and SiOC ceramics – structure and energetics at the nanoscale

The complex nanodomain structure and energetic features of polymer-derived SiCN and SiOC ceramics have been investigated by multinuclear 1D and 2D solid-state NMR, micro-Raman, SAXS, XRD, HRTEM and oxidative high temperature oxide melt solution calorimetry, respectively. Structural models consistent with all these lines of evidence put emphasis on interconnected fractal domains stabilized by the mixed-bond-structure and interface bonding between nanodomains. The structural and thermodynamic role of hydrogen in the stabilization of interfacial bonding between the nanodomains in polysilylcarbodiimide-derived SiCN is reported as well.