The analytical chemistry of phosphorus and silicon with particular reference to atomic absorption spectroscopy

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Frog foams and natural protein surfactants

Foams and surfactants are relatively rare in biology because of their potential to harm cell membranes and other delicate tissues. However, in recent work we have identified and characterized a number of natural surfactant proteins found in the foam nests of tropical frogs and other unusual sources. These proteins, and their associated foams, are relatively stable and bio-compatible, but with intriguing molecular structures that reveal a new class of surfactant activity. Here we review the structures and functional mechanisms of some of these proteins as revealed by experiments involving a range of biophysical and biochemical techniques, with additional mechanistic support coming from more recent site-directed mutagenesis studies

Aqueous solubilization of C60 fullerene by natural protein surfactants, latherin and ranaspumin-2

C60 fullerene is not soluble in water and dispersion usually requires organic solvents, sonication or vigorous mechanical mixing. However, we show here that mixing of pristine C60 in water with natural surfactant proteins latherin and ranaspumin-2 (Rsn-2) at low concentrations yields stable aqueous dispersions with spectroscopic properties similar to those previously obtained by more vigorous methods. Particle sizes are significantly smaller than those achieved by mechanical dispersion alone, and concentrations are compatible with clusters approximating 1:1 protein:C60 stoichiometry. These proteins can also be adsorbed onto more intractable carbon nanotubes. This promises to be a convenient way to interface a range of hydrophobic nanoparticles and related materials with biological macromolecules, with potential to exploit the versatility of recombinant protein engineering in the development of nano-bio interface devices. It also has potential consequences for toxicological aspects of these and similar nanoparticles

Resonance assignments for latherin, a natural surfactant protein from horse sweat

Latherin is an intrinsically surfactant protein of ~23 kDa found in the sweat and saliva of horses. Its function is probably to enhance the translocation of sweat water from the skin to the surface of the pelt for evaporative cooling. Its role in saliva may be to enhance the wetting, softening and maceration of the dry, fibrous food for which equines are adapted. Latherin is unusual in its relatively high content of aliphatic amino acids (~25 % leucines) that might contribute to its surfactant properties. Latherin is related to the palate, lung, and nasal epithelium carcinoma-associated proteins (PLUNCs) of mammals, at least one of which is now known to exhibit similar surfactant activity to latherin. No structures of any PLUNC protein are currently available. 15N,13C-labelled recombinant latherin was produced in Escherichia coli, and essentially all of the resonances were assigned despite the signal overlap due to the preponderance of leucines. The most notable exceptions include a number of residues located in an apparently dynamic loop region between residues 145 and 154. The assignments have been deposited with BMRB accession number 19067

The structure of latherin, a surfactant allergen protein from horse sweat and saliva

Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting microbial biofilms. How these proteins work as surfactants while remaining soluble and cell membrane-compatible is not known. Nor have their structures previously been reported. We have used protein nuclear magnetic resonance spectroscopy to determine the conformation and dynamics of latherin in aqueous solution. The protein is a monomer in solution with a slightly curved cylindrical structure exhibiting a ‘super-roll’ motif comprising a four-stranded anti-parallel β-sheet and two opposing α-helices which twist along the long axis of the cylinder. One end of the molecule has prominent, flexible loops that contain a number of apolar amino acid side chains. This, together with previous biophysical observations, leads us to a plausible mechanism for surfactant activity in which the molecule is first localized to the non-polar interface via these loops, and then unfolds and flattens to expose its hydrophobic interior to the air or non-polar surface. Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported. Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far

Diversity in the structures and ligand binding sites of nematode fatty acid and retinol binding proteins revealed by Na-FAR-1 from Necator americanus

Fatty acid and retinol binding proteins (FARs) comprise a family of unusual α-helix rich lipid binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein (Ce-FAR-7) is from a subfamily of FARs that does not appear to be important at the host-parasite interface. We have therefore examined Na-FAR-1 from the blood-feeding intestinal parasite of humans, Necator americanus . The three dimensional structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, respectively, reveals an a-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand binding cavity and an additional C-terminal a-helix. Titration of apo -Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein:ligand complexes can be formed. Na-FAR-1, and possibly other FARs, may have a wider repertoire for hydrophobic ligand binding, as confirmed here by our finding that a range of neutral and polar lipids co-purify with the bacterial recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male

1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

Na-FAR-1 is an unusual -helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.Instituto de Investigaciones Bioquímicas de La Plat

Extremely Red Objects from the Hubble Space Telescope NICMOS Parallel Imaging Survey

We present a catalog of extremely red objects (EROs) discovered using the Hubble Space Telescope Near Infrared Camera and Multi-Object Spectrometer (NICMOS) parallel imaging database and ground-based optical follow-up observations. Within an area of 16 arcmin^2, we detect 15 objects with R-F160W > 5 and F160W 6. Our objects have F110W-F160W colors in the range 1.3–2.1, redder than the cluster elliptical galaxies at z ~ 0.8 and nearly 1 mag redder than the average population selected from the F160W images at the same depth. In addition, among only 22 NICMOS pointings, we detected two groups or clusters in two fields; each contains three or more EROs, suggesting that extremely red galaxies may be strongly clustered. At bright magnitudes with F160W < 19.5, the ERO surface density is similar to what has been measured by other surveys. At the limit of our sample, F160W = 21.5, our measured surface density is 0.94 ± 0.24 arcmin^(-2). Excluding the two possible groups or clusters and the one apparently stellar object reduces the surface density to 0.38 ± 0.15 arcmin^(-2)

Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten β-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst β-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes