BACKBONE STRUCTURE AND DYNAMICS OF A HEMOLYMPH PROTEIN FROM THE MEALWORM BEETLE TENEBRIO-MOLITOR

Pheromones play a vital role in the survival of insects and are used f or chemical communication between members of the same species by their olfactory system. The selection and transportation of these lipophili c messengers by carrier proteins through the hydrophilic sensillum lym ph in the antennae toward their membrane receptors remains the initial step for the signal transduction pathway, A moderately abundant 12.4 kDa hydrophilic protein present in hemolymph from the mealworm beetle Tenebrio molitor is similar to 38% identical to a family of insect phe romone-binding proteins, The backbone structure and dynamics of the 10 8-residue protein have been characterized using three-dimensional H-1- N-15 NMR spectroscopy, combined with N-15 relaxation and H-1/D exchang e measurements. The secondary structure, derived from characteristic p atterns of dipolar connectivities between backbone protons, secondary chemical shifts, and homonuclear three-bond J(HNH alpha) coupling cons tants, consists of a predominantly disordered N-terminus from residues 1 to 10 and six alpha-helices connected by four 4-7 residue loops and one beta-hairpin structure, The up-and-down arrangement of alpha-heli ces is stabilized by two disulfide bonds and hydrophobic interactions between amphipathic helices, The backbone dynamics were characterized by the overall correlation time, order parameters, and effective corre lation times for internal motions. Overall, a good correlation between secondary structure and backbone dynamics was found, The N-15 relaxat ion parameters T-1 and T-2 and steady-state NOE values of the six alph a-helices could satisfactorily fit the Lipari-Szabo model, In agreemen t with their generalized order parameters (>0.88), residues in helical regions exhibited restricted motions on a picosecond time scale. The stability of this highly helical protein was confirmed by thermal dena turation studies.