期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2005
卷号：102
期号：15
页码：5386-5391
DOI：10.1073/pnas.0501447102
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 {micro}M-1{middle dot}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 {approx}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 (BmorPBP{Delta}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.
关键词：Bombyx mori pheromone-binding protein ; bombykol ; effect of C terminus on pheromone release ; fast uptake of pheromone and delivery ; mutated pheromone-binding proteins
