Millisecond-time-scale controlled freeze-quench for solute-intermediate analysis by solid-state NMR.

The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo.

The motional dynamics of labile Na atoms within the large channels (Na6 sites) of the zeolite-like Ge-framework in Na1–xGe3+z prevent a standard relative quantification of the two different Na sites (Na6 and Na5) by solid-state NMR at room-temperature (RT). Using low-temperature (−135 °C) 23Na solid-state MAS NMR, we demonstrate that the dynamics can be effectively frozen out, facilitating NMR characterization of the electronic environment of these sites as well as quantification of the Na6/Na5 ratio. By combining the 23Na NMR data with the atomic ratio Na/Ge = 0.25 ± 0.02 from PXRD, we obtain a composition Na1–0.28Ge3+0.13 (or Na0.72Ge3.13) for the specimen studied. The results illustrate a potential pitfall of quantitative analysis based on MAS NMR of quadrupolar nuclei in solids when significant atomic motion is present. Specifically, the absence of satellite transition (ST) spinning sidebands (ssbs) in the MAS NMR spectrum does not necessarily validate the assumption that the ST intensity is fully averaged into the central transition (CT). For the case of the Na6 sites for the present Na1–xGe3+z sample, reorientational motion at RT results in loss of ST ssbs; however, the motion is not sufficiently fast to fully average the ST intensity into the CT, and quantitative analysis based on such an assumption can lead to erroneous conclusions. The complete Na6 ssb manifold could be observed and the correct ratio Na6/Na5 = 1.9 ± 0.1 obtained only after reducing the sample temperature to −135 °C.

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.

The effect of dissolved impurity on calcium phosphate nucleation in supersaturated medium

H heteronuclear dipolar coupling in dilute membrane proteins oriented in hydrated anddielectrically lossy lipid environments. The system employed a 800 MHz narrow-bore magnet. A solenoid coilstrip shield was used to reduce deleterious RF sample heating by minimizing the conservative electric fieldsgenerated by the double-tuned resonator at high magnetic fields. The probe's design, construction, andperformance in solid-state NMR experiments at high magnetic fields are described here. Such high-resolutionsolid-state NMR spectroscopic analysis of static oriented samples in hydrated phospholipid bilayers or bicellescould aid the structural analysis of dilute biological membrane proteins.Key Words : Solid-state NMR probe, Low- e, Probe design, Membrane proteins, BicellesIntroductionMembrane proteins are important in various essentialsystems and regulatory processes, ranging from the com-plicated network of cellular communications to the meta-bolism and breaking down of unwanted substances in thehuman body. Hundreds of diseases involve the mis-foldingor mis-assembling of integral membrane proteins and over60% of all drug molecules target membrane proteins.Despite their importance, these protein's intrinsic structure-function relationships are not understood as their three-dimensional structures have not been fully elucidated. Thestudy of membrane proteins by current methods is hamperedby the need of a native bilayer environment, which is diffi-cult using most biophysical techniques. They are notoriouslydifficult to crystallize for X-ray crystallography and theirslow reorientation rates when combined with lipids or deter-gents hinder the use of conventional solution NMR (NuclearMagnetic Resonance) techniques. Solid-state NMR spectroscopy is a relatively new meansof investigating the structures of solid biological samples.

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization-based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

Modulation of TNAP activity and apatite formation in biomimetic matrix vesicles studied by 31P solid-state NMR.

BackgroundResearchers are looking for biomimetic mineralization of ena/mel to manage dental erosion. This study evaluated biomimetic mineralization of demineralized enamel induced by a synthetic and self-assembled oligopeptide amphiphile (OPA).ResultsThe results showed that the OPA self-assembled into nano-fibres in the presence of calcium ions and in neutral acidity. The OPA was alternately immersed in calcium chloride and sodium hypophosphate solutions to evaluate its property of mineralization. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed nucleation and growth of amorphous calcium phosphate along the self-assembled OPA nano-fibres when it was repetitively exposed to solutions with calcium and phosphate ions. Energy dispersive spectrometry (EDS) confirmed that these nano-particles contained calcium and phosphate. Furthermore, electron diffraction pattern suggested that the nano-particles precipitated on OPA nano-fibres were comparable to amorphous calcium phosphate. Acid-etched human enamel slices were incubated at 37°C in metastable calcium phosphate solution with the OPA for biomimetic mineralization. SEM and X-ray diffraction indicated that the OPA induced the formation of hydroxyapatite crystals in organized bundles on etched enamel. TEM micrographs revealed there were 20–30 nm nano-amorphous calcium phosphate precipitates in the biomimetic mineralizing solution. The particles were found separately bound to the oligopeptide fibres. Biomimetic mineralization with or without the oligopeptide increased demineralized enamel microhardness.ConclusionsA novel OPA was successfully fabricated, which fostered the biomimetic mineralization of demineralized enamel. It is one of the primary steps towards the design and construction of novel biomaterial for future clinical therapy of dental erosion.

Rapid cooling to a solid state allows intermediates in chemical and biomolecular processes that occur in solution near room temperature to be trapped for subsequent measurements by magnetic resonance spectroscopies, electron microscopy, or other techniques. In time-resolved solid state nuclear magnetic resonance and rapid freeze-quench electron paramagnetic resonance studies, solutions are typically frozen by spraying into a cold bath or onto a cold metal surface. Although simulations suggest freezing on millisecond or submillisecond time scales, direct experimental measurements of cooling rates have been elusive. Here, we describe a method for quantification of rapid cooling rates based on measurements of temperature-dependent photoluminescence from thioflavin T (ThT). In our experiments, a jet of ThT solution in glycerol/water, with 10.8 m/s jet velocity and 30 μm diameter, freezes on a cold, rotating copper surface. Images of ThT photoluminescence on the copper surface indicate that the cooling rate of the solution increases linearly with the surface velocity over the 0.45-6.2 m/s range. At surface velocities greater than 3.8 m/s, the time to cool from 300 to 260 K or from 300 to 230 K is less than 100 μs or less than 700 μs. The experimental results do not agree quantitatively with calculations in which a layer of glycerol/water cools by thermal conduction when suddenly brought in contact with a cold copper surface. Discrepancies between experimental results and simplistic calculations illustrate the importance of direct measurements of cooling rates.

In idiopathic recurrent urolithiasis (IRCU) calcium oxalate and calcium phosphate are components of stones. It is not sufficiently known whether in urine the nucleation (liquid-solid transition) of each salt requires a different environment, if so which environment, and whether there is an impact on stone formation. Nucleation was induced by in vitro addition of oxalate or calcium to post-test meal load whole urine of male stone patients (n=48), showing normal daily and baseline fasting oxaluria. The maximally tolerated (until visible precipitates occur) concentration of oxalate (T-Ox) or calcium (T-Ca) was determined; additionally evaluated were other variables in urine, including total, complexed and free citrate (F-Cit), protein (albumin, non-albumin protein) and the clinical intensity (synonymous metabolic activity; MA) of IRCU. In the first of three trials the accumulation of substances in stone-forming urine was verified (trial-V); in the second (clinical trial 1) two strata of T-Ox (Low, High) were compared; in the third (clinical trial 2) IRCU patients (n=27) and a control group (n=13) were included to clarify whether in stone-forming urine the first crystal formed was calcium oxalate or calcium phosphate, and to identify the state of F-Cit. T-Ox was studied at the original pH (average < 6.0), T-Ca at prefixed pH 6.0; the precipitates were subjected to electron microscopy and element analysis. Trial-V: Among the urinary substances accumulating at the indicated pHs were calcium, oxalate and phosphate, and the crystal-urine ratios were compatible with the nucleation of calcium oxalate, calcium-poor and calcium-rich calcium phosphate; citrate, protein and potassium also accumulated. Clinical trial 1: the two strata exhibited an inverse change of T-Ox and T-Ca, the ratio T-Ox/T-Ca and MA. The initial (before induction of Ox or Ca excess) supersaturation of calcium oxalate and brushite were unchanged, with the difference of proteinuria being borderline. Several correlations were significant (p < or = 0.05): urine pH with citrate and volume, protein with volume and MA, T-Ox with T-Ca and MA. Clinical trial 2: in patients with reduced urine volume and moderate urine calcium excess, the first precipitate appeared to be calcium oxalate, followed by amorphous calcium phosphate. Conversely, when the calcium excess was extreme, calcium-rich hydroxyapatite developed, followed by calcium oxalate; F-Cit, not total and complexed citrate, was decreased in IRCU vs. male controls; F-Cit rose pH-dependently, and the ratio F-Cit at original pH vs. F-Cit at pH 6.0 correlated inversely with the nucleation index T-Ox/T-Ca; MA correlated inversely with the ratio F-Cit at pH 6.0, respectively, original pH, but directly with the urinary albumin/non-albumin protein ratio. In summary 1) to study calcium oxalate and calcium phosphate nucleation in whole urine of IRCU patients is feasible; 2) at this crystallization stage the two substances, dominant in calcium stones, appear intimately linked, 3) in stone-forming urine, calcium phosphate may be ubiquitously present, likely as particles < 0.22 microm; 4) together with co-precipitation of calcium oxalate and calcium phosphate, low F-Cit and alteration of proteinuria may act in concert and accelerate stones.

A study of barrier film growth on aluminium in solutions of film-promoting and aggressive ions using secondary ion mass spectrometry

Numerous ionic crystals are known in which both cations and anions possess considerable mobility in the solid state. During the past decade, there has been considerable controversy about the question of whether cationic and anionic motion can be dynamically coupled in such materials. This issue has been studied recently on the plastic crystalline material high-temperature (HT-) Na3PO4, which forms from the low-temperature modification in a first-order phase transition at a temperature near 600 K. In the present study, the dynamics of the low-temperature phase have been characterized comprehensively by complementary NMR methods. Temperature-dependent 17O NMR line shape analyses indicate that the phosphate ions undergo 3-fold rotation on the time scale of milliseconds. There appears to be one preferred axis of rotation, however. Variable-temperature 23Na and 31P NMR spectra reveal further that the sodium cations exhibit considerable mobility. Both anionic and cationic motion appear to be jointly thermally activated and are characterized by correlation times of comparable magnitude. At temperatures about 70 K below the phase transition, diffuse diffraction peaks observed in X-ray powder diffraction data indicate the appearance of local clusters possessing the symmetry of the high-temperature phase. The strongly increased thermal volume expansion coefficient and the observation of excess specific heat within this temperature range suggest that both the cations and the anions exhibit strongly accelerated dynamics within these domains. The number of nuclei contributing to these domains are quantified on the basis of 17O and 23Na NMR line shape and nutation analyses. The combined experimental evidence suggests strong dynamic coupling between anion and cation motion in low-temperature (LT-) Na3PO4.

BackgroundPlant cell walls are made of a complex network of interacting polymers that play a critical role in plant development and responses to environmental changes. Thus, improving plant biomass and fitness requires the elucidation of the structural organization of plant cell walls in their native environment. The 13C-based multi-dimensional solid-state nuclear magnetic resonance (ssNMR) has been instrumental in revealing the structural information of plant cell walls through 2D and 3D correlation spectral analyses. However, the requirement of enriching plants with 13C limits the applicability of this method. To our knowledge, there is only a very limited set of methods currently available that achieve high levels of 13C-labeling of plant materials using 13CO2, and most of them require large amounts of 13CO2 in larger growth chambers.ResultsIn this study, a simplified protocol for 13C-labeling of plant materials is introduced that allows ca 60% labeling of the cell walls, as quantified by comparison with commercially labeled samples. This level of 13C-enrichment is sufficient for all conventional 2D and 3D correlation ssNMR experiments for detailed analysis of plant cell wall structure. The protocol is based on a convenient and easy setup to supply both 13C-labeled glucose and 13CO2 using a vacuum-desiccator. The protocol does not require large amounts of 13CO2.ConclusionThis study shows that our 13C-labeling of plant materials can make the accessibility to ssNMR technique easy and affordable. The derived high-resolution 2D and 3D correlation spectra are used to extract structural information of plant cell walls. This helps to better understand the influence of polysaccharide-polysaccharide interaction on plant performance and allows for a more precise parametrization of plant cell wall models.

4 SO ions and OH ions play important roles in hydrothermal synthesis of Ni(OH)2 nanostructures with only inorganic nickel salt and NaOH. The whole process could be divided into 2 parts, the preparation of Ni(OH)2 gel and its hydrothermal reaction. According to our analysis, the former one is an ionic-reaction which produces amorphous Ni(OH)2. It is the later one when Ni(OH)2 gel crystallizes that dominates the polymorph and morphology of final products. The influence of OHon morphologies of Ni(OH)2 has been studied by changing the ratio of n( 4 SO )/n(OH -). To discuss the effect of 2 4 SO ions in hydrothermal system on crystal growth, we wash the gel which is prepared by NiCl2 and NaOH with the mixture solution of Na2SO4 and NiSO4 for different times, so that Cl ions in the gel solution could be replaced by 4 SO ions in varying degrees. Then the nanosized Ni(OH)2 products is prepared by hydrothermal treatment. Also, the samples were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). TEM results show that there will be 2 main nanostructures in different hydrothermal conditions: nanowires and nanosheets. XRD and FTIR results reveals that the nanowires is Ni(SO4)0.3(OH)1.4, one kind of α-Ni(OH)2, and the nanosheet is β-Ni(OH)2. We finally got a conclusion that low amount of OH and pure 4 SO environment is advantageous to synthesis of α-Ni(OH)2 nanowires. Since α-Ni(OH)2 is hydroxyl-deficient and consists of stacked Ni(OH)2-x layers toward which the 4 SO ion has a higher affinity than other ions such as Cl and 3 NO , the 4 SO ion is able to enter its interlayers. The structure is stabilized due to the strong coordination bonds between 4 SO and Ni + in Ni(OH)2-x layers. Also, free Ni ions in solution would be linked to intercalated 4 SO through coordination bonds, so that new Ni(OH)2-x layers could form constantly and stack along the c-axis. As a result, there will be a preferential growth in the [001] direction. However, this process is not likely to continue when there is high amount of OH-, which may cause high Coulomb repulsion along the [001] direction.