Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein.
The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.
Touhara
K
K
Department of Chemistry, State University of New York, Stony Brook 11794-3400.
Prestwich
G D
GD
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003979:Diazonium Compounds / Q000378:metabolism*; D005204:Farnesol / Q000031:analogs & derivatives* / Q000378:metabolism; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism; D007814:Larva; D008961:Models, Structural; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010782:Photolysis; D011487:Protein Conformation
D000345:Affinity Labels; D002352:Carrier Proteins; D003979:Diazonium Compounds; D019476:Insect Proteins; D007605:Juvenile Hormones; D010446:Peptide Fragments; C035606:juvenile hormone-binding protein, insect; D005204:Farnesol; C066480:epoxyhomofarnesyl diazoacetate