Key disulfide bonds in an insect hormone binding protein: cDNA cloning of a juvenile hormone binding protein of Heliothis virescens and ligand binding by native and mutant forms.
The hemolymph juvenile hormone binding protein (JHBP) from the early fifth instar larvae of Heliothis virescens (Lepidoptera, Noctuidae) has been purified, and three cDNA clones for this protein have been isolated from a fat body cDNA library constructed in bacteriophage lambda ZAP XR. The deduced amino acid sequence of the full-length clone predicts a mature protein consisting of 224 residues, a molecular mass of 24,976 Da, and a pI of 5.29. Comparison of the amino acid sequence to that of the previously described JHBP from Manduca sexta shows 51% overall identity with highly conserved N- and C-terminal regions. One of the three clones bound photoactivatable analogs of juvenile hormones with much lower affinity than the other two. This clone had Phe150 in place of the expected Cys150 conserved in other JHBP clones. The F150C mutant of this clone regained native binding affinity. For native Hvir-JHBP, the affinity for [3H]JH I was lower under reducing conditions (87 nM) relative to a 40 nM affinity under nonreducing conditions. The importance of pairs of Cys residues was addressed by preparing Cys to Ala mutants at each site. Expressed proteins were tested for binding affinity by photoaffinity labeling with tritium-labeled JH analogs and by binding assays using (10R,11S)-[3H]JH I. Curiously, the C150A mutant retained full activity, implying that the aberrant C150F was dysfunctional due to steric hindrance rather than to a missing disulfide linkage. Likewise, C29A and C194A had binding affinities unchanged from that of the full-length wild-type clone.(ABSTRACT TRUNCATED AT 250 WORDS)
Wojtasek
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Department of Chemistry, State University of New York at Stony Brook 11794-3400, USA.
Prestwich
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GD
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004220:Disulfides; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism*; D007814:Larva / Q000737:chemistry; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry
D000345:Affinity Labels; D002352:Carrier Proteins; D018076:DNA, Complementary; D004220:Disulfides; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect