Kinetics and molecular properties of pheromone binding and release.
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 microM(-1).s(-1). With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is approximately equal to 1 ms. A pH-dependent conformational change (BmorPBP(B) --> BmorPBP(A)), associated with the release of pheromone, is a first-order reaction (k = 74.1 +/- 0.32 s(-1); t(1/2), 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 (BmorPBPDeltaP129-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.
Leal
Walter S
WS
Maeda-Duffey Laboratory, Department of Entomology, University of California, Davis, CA 95616, USA. wsleal@ucdavis.edu
Chen
Angela M
AM
Ishida
Yuko
Y
Chiang
Vicky P
VP
Erickson
Melissa L
ML
Morgan
Tania I
TI
Tsuruda
Jennifer M
JM
D000818:Animals; D012831:Bombyx; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005233:Fatty Alcohols / Q000737:chemistry* / Q000378:metabolism*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008958:Models, Molecular; D010675:Pheromones / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D005233:Fatty Alcohols; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010675:Pheromones; C403094:pheromone-binding protein, Bombyx; C006787:bombykol; D014364:Tryptophan