Revisiting the Specificity of Mamestra brassicaeand Antheraea polyphemus Pheromone-binding Proteins with a Fluorescence Binding Assay

Campanacci, Valérie; Krieger, Jürgen; Bette, Stefanie; Sturgis, James N.; Lartigue, Audrey; Cambillau, Christian; Breer, Heinz; Tegoni, Mariella

doi:10.1074/jbc.m100713200

Cited by 197 publications

(197 citation statements)

References 43 publications

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“…3, A-C). The K d values determined were 0.90, 1.60, and 0.35 M, respectively, in the range of values observed for PBP-pheromone complexes (31,33).…”

Section: Resultsmentioning

confidence: 86%

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

Lartigue¹,

Campanacci²,

Roussel³

et al. 2002

Journal of Biological Chemistry

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161

187

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Two classes of highly soluble and very abundant proteins of ϳ150 amino acids have been detected in sensilla of Lepidoptera, both containing 6 conserved cysteines forming three disulfide bridges. The first class, that of GOBPs, 1 is equally distributed in both sexes, whereas the second class, that of PBPs, is mainly present in males (1). A third class of small proteins (average M r 13,000) has been identified in antennae from Drosophila melanogaster and in antennae and several sensorial organs (tarsi, labrum) from a wide range of species of the insect order (2-11). These proteins have been proposed to be involved in CO 2 detection (3), in chemical signal transmission in regenerating legs (5), or in chemo-perception (either olfaction or taste (9, 12)), and they were therefore called chemosensory proteins (CSPs). CSPs are shorter (110 -115 amino acids) than PBP or GOBP, contain only 4 conserved cysteines forming two disulfide bridges (9), and share no sequence homology with them.

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“…3, A-C). The K d values determined were 0.90, 1.60, and 0.35 M, respectively, in the range of values observed for PBP-pheromone complexes (31,33).…”

Section: Resultsmentioning

confidence: 86%

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

Lartigue¹,

Campanacci²,

Roussel³

et al. 2002

Journal of Biological Chemistry

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161

187

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“…A higher value (K D 1,100 nM) at pH 8, determined by a tryptophan fluorescence-based binding assay, has been reported (14). In contrast to fluorescence assays, the cold binding assay allows accurate measurements of the concentrations of free and bound ligand (by GC) and protein (by LC-ESI͞MS), thus permitting a more accurate determination of dissociation constants.…”

Section: Resultsmentioning

confidence: 99%

“…The procedure allows the evaluation of binding by direct detection of the ligand but requires larger amounts of protein than the quantitative native gel assay (12). Our cold binding assay was extensively evaluated by using BmorPBP and bombykol, whose binding ability at high pH (and lack of binding at low pH) has been well documented (8,13,14). Results of the cold binding assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinetics and molecular properties of pheromone binding and release

Leal

Chen

Ishida

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

164

139

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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...

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“…The K d values and the standard errors were estimated by non-linear regression using Prism 3.02 (GraphPad software, Inc) following procedures already described [19].…”

Section: Tryptophan Fluorescence Quenching Studiesmentioning

confidence: 99%

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

et al. 2006

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Lipocalins, a widespread multifunctional family of small proteins (15-25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K d range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K d range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.

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Revisiting the Specificity of Mamestra brassicaeand Antheraea polyphemus Pheromone-binding Proteins with a Fluorescence Binding Assay

Cited by 197 publications

References 43 publications

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

Kinetics and molecular properties of pheromone binding and release

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

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