Kinetics and molecular properties of pheromone binding and release

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Abstract
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 μM–1·s–1. With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromonein vivois ≈1 ms. A pH-dependent conformational change (BmorPBPB→ BmorPBPA), 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 (KD= 105 nM) at pH 7 but low affinity (KD= 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBPΔP129-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.