In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes

The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immunohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its physiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradients emanating from the hemolymph side of the midgut in semi-intact mosquito larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS). Large H+ concentration gradients, with highest concentrations close to the basal membrane (outward [H+] gradients), were found in the anterior midgut, whereas much smaller gradients, with concentrations lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut. Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individuals of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6 h, the maximum duration of the recordings. Bafilomycin A1 (10(-5), 10(-7 )mol x l(-1)) on the hemolymph side greatly diminished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut. These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic H+ V-ATPase energizes luminal alkalization in the anterior midgut of larval mosquitoes

Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: A well-ordered β-sheet core amidst structural heterogeneity.

GrEASy fibrils: Hydrophobins are fungal proteins that assemble into an amphipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid

Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state

Characterization of soil organic matter in aggregates and size-density fractions by solid state C-13 CPMAS NMR spectroscopy.

Understanding the changes in soil organic matter (SOM) composition during aggregate formation is crucial to explain the stabilization of SOM in aggregates. The objectives of this study were to investigate (i) the composition of SOM associated with different aggregates and size-density fractions and (ii) the role of selective preservation in determining the composition of organic matter in aggregate and size-density fractions. Surface soil samples were collected from an Alfisol on the Northern Tablelands of NSW, Australia with contrasting land uses native pasture, crop-pasture rotation and woodland. Solid state 13C cross-polarization and magic angle spinning (CPMAS) Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the SOM composition in macroaggregates (250-2000 µm), microaggregates (53-250 µm), and <53 µm fraction. The chemical composition of light fraction (LF), coarse particulate organic matter (cPOM), fine particulate organic matter (fPOM) and mineral associated soil organic matter (mSOM) were also determined. The major constituent of SOM of aggregate size fractions was O-alkyl carbon, which represented 44-57% of the total signal acquired, whereas alkyl carbon contributed 16-27%. There was a progressive increase in alkyl carbon content with decrease in aggregate size. Results suggest that SOM associated with <53 µm fraction was at a more advanced stage of decomposition than that of macroaggregates and microaggregates. The LF and cPOM were dominated by O-alkyl carbon while alkyl carbon content was high in fPOM and mSOM. Interestingly, the relative change in O-alkyl, alkyl and aromatic carbon between aggregates and SOM fractions revealed that microbial synthesis and decomposition of organic matter along with selective preservation of alkyl and aromatic carbon plays a significant role in determining the composition of organic matter in aggregates

Access to side-chain carbon information in deuterated solids under fast MAS through non-rotor-synchronized mixing.

We demonstrate the accessibility of aliphatic 13C side chain chemical shift sets for solid-state NMR despite perdeuteration and fast MAS using isotropic, non-rotor-synchronized 13C-13C mixing. Combined with amide proton detection, we unambiguously and sensitively detect whole side chain to backbone correlations for two proteins using around 1 mg of sample