Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an ''on'' rate of 0.068 ؎ 0.01 M ؊1 ⅐s ؊1 . With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is Ϸ1 ms. A pH-dependent conformational change (BmorPBP B 3 BmorPBP A ), associated with the release of pheromone, is a first-order reaction (k ‫؍‬ 74.1 ؎ 0.32 s ؊1 ; t1/2, 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K D ‫؍‬ 105 nM) at pH 7 but low affinity (K D ‫؍‬ 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBP⌬P129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.Bombyx mori pheromone-binding protein ͉ bombykol ͉ effect of C terminus on pheromone release ͉ fast uptake of pheromone and delivery ͉ mutated pheromone-binding proteins F or many insects, small-molecule signals communicate the availability of food, the presence of friends and foes, and the readiness to mate. In general, mate-finding is an essential prerequisite for exploring insects' enormous reproductive potential and, consequently, leads to their domination of the terrestrial world. To advertise their readiness to mate, female moths, for example, produce and release sex pheromones. Only minute amounts are released, so as to avoid chemical conspicuousness. Once released, the chemical signals are diluted in the environment and mixed with a myriad of physiologically irrelevant compounds, including pheromones from other species. Even though each species communicates with a specific pheromonic language, males can detect the low-level signals from conspecific females because of a highly developed olfactory system. To find females and successfully reproduce, males may have to make long-distance, odorant-oriented flights. Navigation requires a highly selective, sensitive, and dynamic sensory system for the detection of pheromones. Given the structure of pheromone plumes (1), insects have only a few milliseconds to reset their detectors while flying in the clean air spaces between pockets of chemical signals.Pheromones are largely hydrophobic compounds, whereas pheromo...

With a protein-based approach, we have identified and cloned the cDNA encoding a chemosensory protein (LhumCSP) in the Argentine ant, Linepithema humile. The open reading frame of the cloned cDNA encoded a signal peptide (20 residues), and a mature protein (pI 4.62) of 106 amino acid residues. The calculated molecular mass (12,453 Da) was in agreement with the molecular mass measured by on-line chromatography-electrospray ionization mass spectrometry (12,448 Da), given the formation of two disulfide bridges. LhumCSP shared sequence similarity with various CSPs, particularly those identified and/or cloned from moth species. Also, LhumCSP showed the hallmark of the chemosensory proteins, i.e., four well conserved cysteine residues. The antennal protein was not detected in non-olfactory tissues (leg and thorax) contrary to a putative pheromone-binding protein isolated from the thorax of the red imported fire ant, Solenopsis invicta. In addition, these findings suggest that, as in Orthopterans and Phasmids, the protein that makes sense in the Argentine ant is not an odorant-binding protein, but rather a chemosensory protein.

No abstract

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.