Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs—gastroliths, readily providing the Ca 2+ needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus . In addition to the known CaCO 3 , chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith “soluble matrix.” The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found < 5  Å from a phosphate (intermolecular C⋯P distance), an interaction that must be mediated by Ca 2+ . The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO 3 . Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

The molecular interface between bioorganics and inorganics plays a key role in diverse scientific and technological research areas including nanoelectronics, biomimetics, biomineralization, and medical applications such as drug delivery systems and implant coatings. However, the physical/chemical basis of recognition of inorganic surfaces by biomolecules remains unclear. The molecular level elucidation of specific interfacial interactions and the structural and dynamical state of the surface bound molecules is of prime scientific importance. In this study, we demonstrate the ability of solid state NMR methods to accomplish these goals. L-[1-(13)C,(15)N]Alanine loaded onto SBA-15 mesoporous silica with a high surface area served as a model system. The interacting alanine moiety was identified as the -NH(3)(+) functional group by (15)N{(1)H}SLF NMR. (29)Si{(15)N} and (15)N{(29)Si}REDOR NMR revealed intermolecular interactions between the alanine -NH(3)(+) and three to four surface Si species, predominantly Q(3), with similar internuclear N...Si distances of 4.0-4.2 A. Distinct dynamic states of the adsorbed biomolecules were identified by (15)N{(13)C}REDOR NMR, indicating both bound and free alanine populations, depending on hydration level and temperature. In the bound populations, the -NH(3)(+) group is surface anchored while the free carboxylate end undergoes librations, implying the carboxylate has small or no contributions to surface binding. When surface water clusters grow bigger with increased hydration, the libration amplitude of the carboxyl end amplifies, until onset of dissolution occurs. Our measurements provide the first direct, comprehensive, molecular-level identification of the bioorganic-inorganic interface, showing binding functional groups, geometric constraints, stoichiometry, and dynamics, both for the adsorbed amino acid and the silica surface.

Major advances over the last few years have facilitated the synthesis of a large variety of meso-only substituted corroles that display interesting catalytic, therapeutic and photophysical properties. This work is the first to study extensively the NMR spectral characteristics of both metallated and non-metallated triarylcorroles in various organic solvents and provide guidelines for easy and reliable assignments of 1D 1H spectra from trends of J coupling constants and chemical shifts. An excellent correlation is found between C=C bond lengths derived from 3J(H,H) values and experimental lengths determined by x-ray crystallography of the same molecules. The nuclear Overhauser effect provides a robust 1D 1H NMR tool for determining the selectivity of electrophilic substitutions. Variable-temperature NMR and isotopic labelling reveal a single preferred tautomerization state and unsymmetric ring orientations at -70 degrees C. The beta-pyrrole protons demonstrate long-range heteronuclear couplings with the coordination core (15N) and with the ortho-19F nuclei of the meso-carbon aryl rings. In sum, application of multinuclear magnetic resonance to corroles and their metal complexes, through the compilation of chemical shifts and J couplings and the recognition of trends therein, provides basic information essential to reliable spectral assignments. Additionally, the conclusions drawn about the structures of corroles and the electron densities at various positions of the corrole macrocycle resulting from the application of high-resolution NMR techniques are of importance to an in-depth understanding of the molecular interactions and processes of this relatively new and rapidly expanding class of compounds.

Background: Acetylxylan esterases are enzymes that remove acetyl groups from the hemicellulolytic polymer xylan. Results: The axe2 gene product in Geobacillus stearothermophilus removes acetyl groups from acetylated xylan and xylosaccharides. Conclusion: Axe2 represents a new serine carbohydrate esterase family. Significance: The findings may provide new routes for the efficient utilization of biomass as a renewable energy source.

No abstract

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of alpha-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K(M(acceptor)), respectively, and a k(cat)/K(M(acceptor)) value of 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.

The backbone conformation of peptides and proteins is completely defined by the torsion angles (φ,ψ,ω) of each amino acid residue along the polypeptide chain. We demonstrate a solid-state NMR method based on heteronuclear distance measurements for determining (φ,ψ) angles. Simple and reliable deuterium phase modulated pulses (PM5) reintroduce dipolar couplings between 2H and a spin-1/2 nucleus. Measuring the 13C i- 1{2H i α} REDOR distance across a peptide bond results in the torsion angle φ i as a consequence of the restricted geometry of the peptide backbone. The 15N i+ 1{2H i α} REDOR distance across a peptide bond defines the torsion angle ψ i . This approach is demonstrated for both the 3-spin X{2H2}REDOR case of glycine and the 2-spin X{2H}REDOR case, represented by l-alanine, using two different tripeptides. It is shown that the technique can handle multiple sample conformations. PM5-REDOR decay curves of the ψ angle show distinctly different behaviors between α-helix and β-sheet backbone conformations.

The first solid state 29Si NMR of a disilyne, that is, RSiSiR, R = Si(CH(SiMe3)2)2(i-Pr) (1) was measured: delta11 = 364 +/- 20; delta22 = 221 +/- 16 and delta33 = -350 +/- 13; CSA = -643 ppm. These measured values as well as calculations for model disilynes strongly support the description of the Si-Si bond in bent disilynes as a triple bond, although with weakened pi-bonds and a reduced bond order of 2.6.