Kinetics and molecular properties of pheromone binding and release

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 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.

Fig. 1.

Association kinetics of bombykol and BmorPBP. (A) Fluorescence decrease upon mixing bombykol and BmorPBP^B at pH 7. The curve was fitted (reduced χ², 0.76) by using two-exponential terms and used to calculate the association rate (k_on). (B) At low pH, the association of bombykol and BmorPBP^A is 10 times slower as indicated by the time course and the kinetic parameters obtained by curve fitting (reduced χ², 0.15) with two-exponential terms.

Fig. 2.

Fluorescence transients of BmorPBP upon changing the pH. (A) The time dependence of the basic to acidic (B → A) conformational change is fitted by one exponential term to generate the observed rate constant (k) (reduced χ², 1.95). (B) The time course of the reverse reaction (A → B) was fitted by two-exponential terms (k₁ and k₂). Note the faster step followed by a second, slower process (reduced χ², 0.59). (Inset) A steep increase in fluorescence intensity in the first 150 ms of the reaction is shown.

Fig. 3.

Bombykol binding to native and truncated PBPs. (A) pH-dependent binding ability. As a negative control, the green leaf volatile (Z)-3-hexenyl acetate did not show significant binding to BmorPBP. The effect of pH on the binding ability of the native conformation of BmorPBP and rescue of binding affinity at low pH in a truncated protein (BmorPBPPΔP129-V142) are shown. (B) Ionic strength and binding ability. Physiologically relevant or extremely high salt concentrations did not affect the binding affinity at high pH and did not rescue binding ability at low pH.

Fig. 4.

Fluorescence emission spectra and binding affinity of native and tryptophan-mutated PBPs at pH 7. (A) Intrinsic fluorescence of BmorPBP, BmorPBPW127F, and BmorPBPW37F with excitation at 280 nm and maximal emission at 335, 334, and 341 nm, respectively. The concentrations of the four proteins were adjusted by LC-ESI/MS. (B) Effect of tryptophan on binding ability. The three tryptophan mutants (BmorPBPW127F, BmorPBPW37F, and BmorPBPW37A) showed binding ability to bombykol nearly identical to that of the native conformation (BmorPBP).