Binding Conformation and Kinetics of Two Pheromone-Binding Proteins from the Gypsy Moth Lymantria dispar with Biological and Nonbiological Ligands

Abstract

Pheromone-binding proteins (PBPs) in insects can bind various substances and selectively deliver the message of a signal molecule to the downstream components of the olfactory system. This can be achieved either through a ligand-specific conformational change of the C-terminal peptide of the PBP or by selectively binding/releasing the ligand. PBP may also act as a scavenger to protect the sensory neurons from saturating at high ligand doses. We have compared two PBPs from the gypsy moth (PBP1 and PBP2) and their truncated forms (TPBPs), which lack the C-terminal peptide, in this study. Stopped-flow kinetics with N-phenyl-1-naphthylamine (NPN) have revealed a diffusion-controlled collisional step, between PBP and NPN, after which the NPN relocates into a hydrophobic environment. This work supports the hypothesis that binding between PBPs and ligands occurs stepwise. With the method of tryptophan fluorescence quenching, we have shown different local conformational changes around Trp 37, induced by different ligands, manifested in changes of both the steric and electronic environment around the residue. Importantly, we have noticed a significant difference in the changes induced by the biological ligand (the pheromone) and nonbiological ligands. Therefore, we hypothesize that PBP may serve a different function in each kinetic step, displaying a unique P.L conformation.

Functional Evolution of Duplicated Odorant-Binding Protein Genes, Obp57d and Obp57e, in Drosophila

“…The competitive binding of fluorescent dyes is widely used to study the interaction between OBPs and small organic compounds in vitro [7], [9], [11], [13], [21], [23], [24]. However, this method could not be applied to Dmel\OBP57d, Dmel\OBP57e, and Dpse\OBP57de because the fluorescent probes, such as N-phenyl-1-naphthylamine (1-NPN) and 1-aminoanthracene (1-AMA), did not bind to these OBPs (data not shown).…”

“…However, this method could not be applied to Dmel\OBP57d, Dmel\OBP57e, and Dpse\OBP57de because the fluorescent probes, such as N-phenyl-1-naphthylamine (1-NPN) and 1-aminoanthracene (1-AMA), did not bind to these OBPs (data not shown). Therefore, the intrinsic fluorescence from tryptophan was used to monitor the interaction [13]. The intensity of the fluorescence from tryptophan varies depending on the surrounding environment.…”

“…Independent models were proposed for the biological role of each OBP in chemosensation. A pheromone-binding protein (PBP) of the gypsy moth Lymantria dispar , PBP2, is thought to function as a scavenger [12], [13]. Another PBP in the silkworm moth Bombyx mori , PBP1, is considered as a transporter for the ligand, bombykol [14].…”

Organization and function of Drosophila odorant binding proteins

“…Many are found in the lymph of olfactory sensilla, where ORN dendrites are located (Shanbhag et al, 2001a). They bind odorants, with different degrees of affinity and selectivity reported for different Obps (Gong et al, 2010; Leal et al, 2005). Within a species, different Obps are expressed in different antennal sensilla (McKenna et al, 1994; Pikielny et al, 1994; Schultze et al, 2013), and some are expressed in the taste system (Galindo and Smith, 2001; Jeong et al, 2013; Pikielny et al, 1994; Shanbhag et al, 2001b) or in larval chemosensory organs (Galindo and Smith, 2001; Park et al, 2000).…”

Analytical methods for kinetic studies of biological interactions: A review

“…2 [52]. This model has also been used to investigate the binding and subsequent change in conformation that occurs for the complex between melittin and Ca 2+ -saturated calmodulin [76], as well as the interaction of a transcriptional activator-DNA complex with a coactivator [77], and the interaction of N -phenyl-1-naphthylamine with pheromone-binding proteins [78]. …”