Esteves Gisele Ferreira, Teles Rozeni Chagas Lima, Cavalcante Nayara Silva, Neves David, Ventura Manuel Mateus, Barbosa João Alexandre Ribeiro Gonçalves, de Freitas Sonia Maria
Laboratório de Biofísica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília-DF, Brazil.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1;63(Pt 12):1087-90. doi: 10.1107/S1744309107056424. Epub 2007 Nov 30.
A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin-BTCI-chymotrypsin ternary complex to 2.7 A resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI-trypsin binary complex (PDB code 2g81) and chymotrypsin (PDB code 4cha) as search models.
采用坐滴气相扩散法，以0.1M HEPES（pH 7.5）、10%（w/v）聚乙二醇6000和5%（v/v）2-甲基-2,4-戊二醇作为沉淀剂，使黑眼豇豆胰蛋白酶和糜蛋白酶抑制剂（BTCI）与胰蛋白酶和糜蛋白酶形成的三元复合物结晶。BTCI是一种从小豇豆种子中分离出的含有83个氨基酸残基的小蛋白质，它能够通过形成稳定的三元复合物同时抑制胰蛋白酶和糜蛋白酶。在低温条件下，从胰蛋白酶-BTCI-糜蛋白酶三元复合物的单晶收集X射线数据，分辨率达到2.7 Å。以BTCI-胰蛋白酶二元复合物（PDB代码2g81）和糜蛋白酶（PDB代码4cha）的晶体结构作为搜索模型，通过分子置换法解析了三元复合物的结构。
