Arthropod repellent interactions with olfactory receptors and ionotropic receptors analyzed by molecular modeling.

The main insect chemoreceptors are olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs). The odorant binding sites of many insect ORs appear to be occluded and inaccessible from the surface of the receptor protein, based on the three-dimensional structure of OR5 from the jumping bristletail (OR5) and a survey of a sample of vinegar fly () OR structures obtained from artificial intellegence (A.I.

Tunnel connects lipid bilayer to occluded odorant-binding site of insect olfactory receptor.

MhOR5, an insect olfactory receptor (OR), has an occluded binding site for the odorant eugenol in both the open and closed states of the ion channel. We used atomistic molecular dynamics simulation (MD) and steered molecular dynamics to examine possible tunnels to the odorant binding site from the protein surface. Four high probability tunnels were identified in the MD results.

Odorant-binding protein from the stable fly (Stomoxys calcitrans) has a high-histidine N-terminal extension that binds transition metals.

The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions.

Antennal Proteome of the Solenopsis invicta (Hymenoptera: Formicidae): Caste Differences in Olfactory Receptors and Chemosensory Support Proteins.

Little is known about the expression pattern of odorant and pheromone transporters, receptors, and deactivation enzymes in the antennae of ants carrying out different tasks. In order to begin filling in this information gap, we compared the proteomes of the antennae of workers and males of the red fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Male ants do not perform any colony work, and their only activity is to leave the nest on a mating flight.

Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum§.

The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons.

Surface Polar Lipids Differ in Male and Female Phlebotomus papatasi (Diptera: Psychodidae).

The polar lipids on the surface of the Old World sand fly, Phlebotomus papatasi (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids.

Surface Polar Lipids Differ in Male and Female (Diptera: Psychodidae).

The polar lipids on the surface of the Old World sand fly, (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids.

Kinetics of lipid mixing between bicelles and nanolipoprotein particles.

Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. Lipids and membrane proteins cannot exchange between NLPs. However, the addition of bicelles opens NLPs and transfers their contents to bicelles, which freely exchange lipids and proteins.

Integral membrane protein fragment recombination after transfer from nanolipoprotein particles to bicelles.

A new method for the measurement of membrane protein oligomer association is described. Two engineered fragments of bacteriorhodopsin, which are known to spontaneously associate in bicelles, were expressed in nanolipoprotein particles (NLPs or nanodiscs) using an Escherichia coli S30 cell-free synthesis system. When separately prepared NLPs containing the fragments were mixed, fragment association did not occur, indicating that the apolipoprotein edge blocks transfer between NLPs.

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles.

To probe structural changes that occur when a membrane protein is transferred from lipid bilayers to SDS micelles, a fragment of bacteriorhodopsin containing transmembrane helical segments A and B was studied by fluorescence spectroscopy, molecular dynamics (MD) simulation, and stopped flow kinetics. In lipid bilayers, Förster resonance energy transfer (FRET) was observed between tyrosine 57 on helix B and tryptophans 10 and 12 on helix A. FRET efficiency decreased substantially when the peptide was transferred to SDS.

Luminescence resonance energy transfer in the cytoplasm of live Escherichia coli cells.

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag.

Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease.

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease.

Helix insertion into bilayers and the evolution of membrane proteins.

Polytopic alpha-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic alpha-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of alpha-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins.

Occurrence of antennal glands in ants.

A previous report of the discovery of exocrine glands in the antennal club of queens and workers of Solenopsis invicta Buren, 1972 left open the question of the extent to which similar glands occur in the Formicidae family. We wanted to know if these antennal glands are unique to Solenopsis, or they are found in a wider taxonomic group. Using scanning electron microscopy, we examined the antennae of 41 ant species.

Buried water molecules in helical transmembrane proteins.

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins.

An unfolding story of helical transmembrane proteins.

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps.

Transmembrane helix-helix association: relative stabilities at low pH.

We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59].

Influenza A (H3N2) outbreak, Nepal.

In July 2004, an outbreak of influenza A (H3N2) was detected at 3 Bhutanese refugee camps in southeastern Nepal. Hemagglutination inhibition showed that approximately 40% of the viruses from this outbreak were antigenically distinct from the A/Wyoming/3/03 vaccine strain. Four amino acid differences were observed in most of the 26 isolates compared with the A/Wyoming/3/2003 vaccine strain.

Apolipophorin-III-like protein expressed in the antenna of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae).

Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence.

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin.

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71).

Structure and distribution of antennal sensilla of the red imported fire ant.

The morphology of the antenna of the red imported fire ant, Solenopsis invicta, was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. The antennae are sexually dimorphic: the worker antenna has porous sensilla on the two distal segments (the antennal club), whereas the clubless male antenna has porous sensilla on all segments past the pedicel. The major type of porous sensilla on both male and female is sensilla tricodea curvata.

Self-association of helical peptides in a lipid environment.

The self-association of two model transmembrane helical peptides, differing in their surface topography, was compared in mixed micelles containing 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) and dimyristoylphosphatidylcholine (DMPC). One peptide, Ac-KKL24KK-amide (L24), has large, rotationally mobile leucine side chains and a relatively rough surface. The other peptide, Ac-KKLLLLLLAALLALLAALLALLLLLLKK-amide (L18A6), has a patch of small alanines on one side of the helix that forms a smooth surface.