MAS-DNP enables NMR studies of insect wings.

NMR is a valuable tool for studying insects. Solid-state NMR has been used to obtain the chemical composition and gain insight into the sclerotization process of exoskeletons. There is typically little difficulty in obtaining sufficient sample quantity for exoskeletons. However, obtaining enough sample of other insect components for solid-state NMR experiments can be problematic while isotopically enriching them is near impossible. This is especially the case for insect wing membranes which is of interest to us. Issues with obtaining sufficient sample are the thickness of wing membranes is on the order of microns, each membrane region is surrounded by veins and occupies a small area, and the membranes are separated from the wing by physical dissection. Accordingly, NMR signal enhancement methods are needed. MAS-DNP has a track record of providing significant signal enhancements for a wide variety of materials. Here we demonstrate that MAS-DNP is useful for providing high quality one-dimensional and two-dimensional solid-state NMR spectra on cicada wing membrane at natural isotopic abundance.

RF field strength control and an envelope detector.

A brief description of a radio-frequency (RF) field strength controller is presented along with an experiment to test the long-term RF field strength stability of a nuclear magnetic resonance (NMR) spectrometer. Active control of RF field strengths may be necessary for quantitative measurements of NMR parameters, especially when experiments require days to run. An envelope detector, easily incorporated in any spectrometer, is also presented. The envelope detector provides an easy way to observe the pulse sequence on an inexpensive oscilloscope and can be useful in troubleshooting.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Description of an rf field-strength controller for solid-state NMR experiments.

Some MAS NMR experiments are sensitive to fluctuations or drifts in rf field strengths. We examine the simple 1H-13C CPMAS experiment and show that the 13C signal intensities vary over time due to drifts in rf field strengths. The causes of the drifts in rf field strengths are studied. Stabilization of the rf field strengths are obtained by using an rf field-strength controller. The controller is a stand-alone unit, which can be easily incorporated in any spectrometer system. Details of the controller, including circuit design, are provided.

Solid-State NMR Study of the Cicada Wing.

Wings of flying insects are part of the cuticle which forms the exoskeleton. The primary molecular components of cuticle are protein, chitin, and lipid. How these components interact with one another to form the exoskeleton is not completely understood. The difficulty in characterizing the cuticle arises because it is insoluble and noncrystalline. These properties severely limit the experimental tools that can be used for molecular characterization. Solid-state nuclear magnetic resonance experiments have been used in the past to characterize the exoskeleton of beetles and have found that chitin and protein make comparable contributions to the molecular matrix. However, little work has been done to characterize the components of the wing, which includes vein and membrane. In this work, solid-state NMR was used to characterize the wing of the 17-year cycle cicada (Magicicada cassini) that appeared in northern West Virginia during the summer of 2016. The NMR results show noticeable differences between the molecular components of the vein and membrane.

Determining the relative orientation between the chemical shift anisotropy and heteronuclear dipolar tensors in static solids by SEDOR NMR.

The measurement of the dipolar interaction between two spins provides the distance between nuclei. A better structural picture emerges when the distance is combined with the orientation of the internuclear vector in the principal axis system of the chemical shift anisotropy tensor. The SEDOR experiment is used on a static sample of alanine to show that the orientation of the vector connecting the nitrogen and carboxylate carbon nuclei can be accurately determined in the CSA PAS of the 13C carboxylate spin.

Adding a lens Improves spinning speed characterization.

Highly stable sample rotation is important in many solid-state NMR experiments. Whether the necessary stability is achieved is not always clear. Typically only an average frequency over some time interval (often relatively long and unknown) is available from the spinning speed controller readout, which is not representative of the short-term variations of instantaneous rotation frequency. The necessity of the relatively slow measurement of spinning speed is a consequence of phase noise in the tachometer, which prevents speed measurement to be both rapid and precise at the same time. We show that adding a lens to the tachometer, without any other changes in the probe, reduces phase noise by nearly an order of magnitude and allows improved measurement of the spinning speed.

Tripeptides on Gold Nanoparticles: Structural Differences between Two Reverse Sequences as Determined by Solid-State NMR and DFT Calculations.

The reverse-sequence peptides CysAlaAla and AlaAlaCys may attach to gold nanoparticles through the thiol group, and they differ primarily by whether the charged amino or the carboxylate group is proximal to the sulfur. Alanine residues in these peptides are not expected to interact significantly with the gold surface and serve to place a large separation between the amino and carboxylate groups. Solid-state NMR experiments and DFT calculations were performed to explore the structural differences between CysAlaAla on gold nanoparticles and AlaAlaCys on gold nanoparticles. It is found that the relative positions between the thiol, amino, and carboxylate groups strongly influences the structures of the peptide-gold systems. CysAlaAla orients parallel to the gold surface in a monolayer fashion, whereas AlaAlaCys forms an interdigitating bilayer-like structure that is oriented upright relative to the gold surface.

Optical lever for monitoring of the magic angle.

Setting the magic angle and accurately maintaining it over a full range of experimental conditions can be a frustrating experience. We present a simple optical device for immediate indication of the stator angle, free of mechanical uncertainties present in typical adjustment mechanisms.

(1)H MAS NMR study of cysteine-coated gold nanoparticles.

(1)H MAS NMR experiments were performed on gold nanoparticles coated with l-cysteine. The experiments show that l-cysteine molecules are zwitterions and support a structural model of cysteine forming two layers. The inner layer is composed of cysteine molecules chemisorbed to the gold surface via the sulfur atom. The outer layer interacts with the chemisorbed layer. The (1)H NMR suggests that the cysteine in the outer layer exhibits large amplitude motion about specific carbon-carbon bonds.

Recollections of REDOR.

Rotational-echo, double-resonance NMR (REDOR) is an experiment designed to measure heteronuclear dipolar couplings in solids and is most often used to obtain structural details in solids. A brief history of its inception and development is presented.

Using heat to control the sample spinning speed in MAS NMR.

A new approach using temperature to control the spinning speed of a sample rotor in magic-angle spinning NMR is presented. Instead of an electro-mechanical valve that regulates the flow of drive gas to control the spinning speed in traditional MAS NMR systems, we use a small heater wire located directly in the stator. The sample spinning speed is controlled very accurately with a surprisingly low heating power of 1 W. Results on a benchtop unit demonstrate the capability of the system.

Oxygen-17 appears only in protein in water-stressed soybean leaves labeled by (17)O2.

We have used a rotational-echo adiabatic-passage double-resonance (13)C{(17)O} solid-state NMR experiment to prove that the glycine produced in the oxygenase reaction of ribulose bisphosphate carboxylase-oxygenase is incorporated exclusively into protein (or protein precursors) of intact, water-stressed soybean leaves exposed to (13)CO(2) and (17)O(2). The water stress increased stomatal resistance and decreased gas exchange so that the Calvin cycle in the leaf chloroplasts was no more than 35% (13)C isotopically enriched. Labeled O(2) levels were sufficient, however, to increase the (17)O isotopic concentration of oxygenase products 20-fold over the natural-abundance level of 0.04%. The observed direct incorporation of glycine into protein shows that water stress suppresses photorespiration in soybean leaves.

Structural analysis of the Gly-rich region in spider dragline silk using stable-isotope labeled sequential model peptides and solid-state NMR.

The local structure of the Gly rich region in synthetic model peptides of spider dragline silk was analyzed with solid-state NMR and no dominant secondary structure was revealed.

A frequency-selective REDOR experiment for an SI2 spin system.

A frequency-selective REDOR experiment is described for SI2 spin systems. The experiment causes the net dipolar dephasing of the S spin to evolve only under the influence of one of the I spins. The experiment is based on a single pair of appropriately phased 90 degrees I-spin pulses, and the I spin causing the S-spin dipolar dephasing is determined by the relative phases between the two 90 degrees pulses. The experiment is demonstrated on a sample of 15N2-L-asparagine.

High field 17O solid-state NMR study of alanine tripeptides.

(17)O chemical shifts of Ala-Ala-Ala, with parallel and anti-parallel beta-sheet structures, are observed using a 930-MHz high-resolution solid-state NMR spectrometer. Ala-Ala-Ala serves as a model sheet-forming peptide because it can be easily prepared as either a parallel or an anti-parallel beta-sheet structure. Spectral differences between the two samples are observed which arise from molecular packing differences between the two sheet structures. DFT chemical shift calculations are performed for the two samples, and the calculated spectra are in good agreement with the experimental spectra. The DFT calculations provide insight into the nature of the chemical shift differences observed between the two sheet structures.

Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR).

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.

Dipolar recoupling of I = 1/2, S = 3/2 spin pairs with SEDOR for static and spinning samples.

Spin-echo, double-resonance (SEDOR) dipolar recoupling experiments are illustrated on an I = 1/2, S = 3/2 spin system for static and spinning samples. An (15)N-(23)Na spin system is used to show that the simple pulse sequence is very effective in causing (15)N dipolar dephasing using either a (23)Na pi/2 recoupling pulse or a long radio-frequency (r.f.) recoupling pulse.

Internuclear (31)P-(51)V distance measurements in polyoxoanionic solids using rotational echo adiabatic passage double resonance NMR spectroscopy.

We report the first results establishing rotational echo adiabatic passage double resonance (REAPDOR) experiments for distance measurements between a spin-1/2 (31P) and spin-7/2 (51V) pair in a series of vanadium-substituted polyoxoanionic solids from the Keggin and Wells-Dawson families. We have quantitatively measured 31P-51V distances in monovanadium substituted K4PVW11O40, 1-K7P2VW17O62, and 4-K7P2VW17O62. Numerical simulations of the experimental data yield very good agreement with the averaged P-W/P-V distances determined from the X-ray diffraction measurements in the same or related compounds. REAPDOR is therefore a very sensitive P-V distance probe anticipated to be especially useful in the absence of long-range order. Our results suggest that REAPDOR spectroscopy could be broadly applicable for interatomic distance measurements in other spin-7/2-spin-1/2 nuclear pairs.