Du G, Ng C S, Prestwich G D
Department of Chemistry, State University of New York at Stony Brook 11794-3400.
Biochemistry. 1994 Apr 26;33(16):4812-9. doi: 10.1021/bi00182a009.
The bacterially expressed recombinant pheromone binding protein (PBP) of Antheraea polyphemus was photoaffinity labeled with (6E,11Z)-[3H]hexadecadienyl diazoacetate, a photoactivatable analog of the naturally occurring acetate pheromone. Radiolabeled peptides were separated from an endoproteinase Lys-C digestion by HPLC and characterized by Edman degradation. The label was exclusively found in the Asp39-Lys58 fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be alpha-helical. The adjoining LAGCAINCLATK fragment, which is highly conserved in PBP sequences, was predicted to be a hydrophobic beta-strand and has been proposed to be important in recognition of the alkadienyl chain. Edman degradation confirmed the location of the covalently attached ligand at Thr44 of the smaller hydrophilic peptide. In addition, the synthesis of the newly identified pheromone component (4E,9Z)-tetradecadienyl acetate and its photoaffinity analog, (4E,9Z)-[3H]tetradecadienyl diazoacetate, is also described. Mapping of PBP photoaffinity labeled by (4E,9Z)-[3H]14:Dza revealed that the hydrophobic region Asp21-Lys38 adjacent to the primary binding domain Asp39-Lys58 contained a second modification site. The 14-carbon odorant molecule thus had two binding positions within the recognition site, while only a single binding position was available to the 16-carbon pheromone.
用（6E,11Z）-[3H]十六碳二烯基重氮乙酸酯（一种天然存在的乙酸酯信息素的光活化类似物）对细菌表达的多音大蚕蛾重组信息素结合蛋白（PBP）进行光亲和标记。通过高效液相色谱（HPLC）从内肽酶Lys-C消化产物中分离出放射性标记的肽段，并通过埃德曼降解法对其进行表征。标记仅在Asp39-Lys58片段中发现。用Arg-C切割该肽（DDYVMTDRLAGCAINCLATK）得到一个单一的放射性标记肽（DDYVMTDR），预计其为α螺旋结构。相邻的LAGCAINCLATK片段在PBP序列中高度保守，预计为疏水β链，并被认为在识别链状二烯基中起重要作用。埃德曼降解法证实了共价连接的配体位于较小亲水性肽的Thr44处。此外，还描述了新鉴定的信息素成分（4E,9Z）-十四碳二烯基乙酸酯及其光亲和类似物（4E,9Z）-[3H]十四碳二烯基重氮乙酸酯的合成。用（4E,9Z）-[3H]14:Dza进行光亲和标记的PBP图谱显示，与主要结合结构域Asp39-Lys58相邻的疏水区域Asp21-Lys38包含第二个修饰位点。因此，14碳的气味分子在识别位点内有两个结合位置，而16碳的信息素只有一个结合位置。
