3A分辨率绿豆胰蛋白酶抑制剂Lys活力碎片—牛胰蛋白酶(MBILF-BTRY)复合物的晶体结构分析和分子模型

用分子置换法成功地解得了MBILF-BTRY的晶体结构,并得到了它的立体结构模型。绿豆胰蛋白酶抑制剂属于絲氨酸蛋白酶抑制剂中结构最复杂的Bowman-Birk型抑制剂。这一类型抑制剂的立体结构以前尚未见报道。MBILF-BTRY复合物晶体的晶胞参数为a=62.99,b=63.54,c=69.70,α=β=γ=90°,空间群为P2_12_12_1,复合物分子量约为27500daltons。用理学电机的Ru-300型转靶X光机及AFC-5型四园衍射仪收集了分辨率为3范围内的独立衍射点5142个。以蛋白质数据库所取得的牛胰蛋白酶分子的座标为     

ACTIVE DOMAINS IN MUNG BEAN TRYPSIN INHIBITOR

In our experimentes, it has been demonstrated that the N-terminal fragment of the mungbean inhibitor could be isolated by affinity chromatography with immobilized trypsin aftertreating the inhibitor with CNB_r and pepsin. The properties of this active fragment have beenstudied. The C-terminal fragment of the hihibitor, however, will lose its activity during CNB_rcleavage because of the scission of the methionyl peptide bond near the active center. It isdeemed desirable to find out a more suitablo method for the cleavage of these two domains,both retaining inhibitory activity. Ths aim has new been achieved by using peptic digestion only, and the active centers ofthese two domains are identified as Lys and Arg by chemical modification. In alkaline pH,these two active fragments could be easily separated by affinity chromatography with immobi-lized trypsin. This amkes it possible to study and compare these two active fragmentsseparately. It is worth pointing out that such a C-terminal fragment with trypsin inhibitory activityhas not yet been reported among the Bowman-Birk inhibitors. The activities of the mung bean trypsin inhibitor decrease to about 50% during thecourse of modification with maleic anhydride and phenylglyoxal respectively. The two activecenters seem to be Lys nd Arg. The mung bean inhibitor could endure peptic digestionwithout any loss of activyty. On Sephadex gel filtration, two smaller active fragments with asize approximately one half of the original molecule are found. At pH 11.4 these two activefragments could be separated from each other by affinity chromatography with immobilizedtrypsin. The active fragment with Lys as the active center could be completely inhibitedwith maleic anhydride and the activity is completely restored after incubation at pH 3.5.This fragmeut consists of two paptide chains with about 35 amino acil residues. The N-termini are found to be Ser and Phe, and the C-termini to be Leu and Met. The activityof the fragment with Arg as the active center could be inhibited completely by phenylglyoxal.This fragment is a single peptide chain with about 27 amino acid residues. The N- and C-terminis are shown to be Asn and Asp respectively.     

绿豆胰蛋白酶抑制剂-猪胰蛋白酶复合物的晶体学研究

本文报道利用微量静置法培养出绿豆胰蛋白酶抑制剂-猪胰蛋白酶复合物可供X射线衍射分析的单晶体。晶体衍射分辨率2.7。晶体学参数测定结果为四方晶系,空间群1422,晶胞参数a=b=122.4(2),c=113.4(2)()。晶体密度1.20g/cm~3,溶剂含量36％,每个结晶学不对称单位含一个复合物分子。     

慈菇蛋白酶抑制剂的抑制特性及其活性中心的探讨

本文采用亲和层析分离纯化了结晶慈菇蛋白酶抑制剂A和B,抑制剂A和B均为双头多功能蛋白酶抑制剂,抑制剂A能同时等当量抑制胰蛋白酶和胰凝乳蛋白酶,对激肽释放酶的抑制作用较弱,抑制剂B能当量抑制2克分子的胰蛋白酶,对激肽释放酶的抑制活力高于抑制剂A,但对胰凝乳蛋白酶的抑制作用远比抑制剂A弱。化学修饰以及胰蛋白酶与胰凝乳蛋白酶对抑制剂A的竞争性结合表明:抑制剂A和B的两个活性中心均为Lys和Arg残基,其中Lys活性中心专一抑制胰蛋白酶,而由Arg活性中心构成的活性区域则表现为多功能,能抑制多种蛋白酶,从抑制剂A和B的结构特征推测,两活性中心应分别为Lys-Ser(44—45)及Arg-Tyr-Lys(76—78),在抑制剂A中还存在一疏水性残基参与对胰凝乳蛋白酶的抑制,此残基位于由Arg活性中心所构成的活性区域中。     

0.3 nm分辨率绿豆胰蛋白酶抑制剂Lys活力碎片—牛胰蛋白酶(MBILF-BTRY)复合物的晶体结构分析和分子模型

Crystal structure of MBILF-BTRY complex was successfully solved by molecular replacement method and its 3-dimensional molecular model was thereby derived. Mung bean trypsin inhibitor belongs to Bowman-Birk inhibitor group, which is by far the most complicated among the ten fundamental groups of serine protease inhibitor. Neither the 3-dimensional structure of Bowman-Birk inhibitor group nor the stereoscopic conformation of its complex with protease has ever been reported. The crystallographic data of MBILF-BTRY complex are found to be a=....     

蛋白质化学的进展

生物高分子核酸与蛋白质是生命活动的最主要的物质基础,前者是生物体遗传信息的主要携带者,后者则是生物体各种活动的主要承担者。生物体中千变万化的活动,从某种意义上也可看成各种活性蛋白质直接或间接活动的结果。例如生命最本质的活动——自     

CRYSTALLIZATION AND PRELIMINARY CRYSTALLOGRAPHIC DATA OF THE MUNG BEAN INHIBITOR

The trypsin inhibitor from mung bean has a molecular weight of 7984, consisting of two active domains and seven disulfide bonds. It belongs to the Bowman-Birk inhibitor. This inhibitor has been crystallized in two crystalline forms either by dialysis or by batch method. One is tetragonal with unit cell of symmetry P4_122(or P4_322) and dimensions a=b=49.21, c=158.07, each asymmetric unit containing three molecules. Another is orthorhombic with unit cell of symmetry P2_12_12 and dimensions a=39.65, b=57.18, c=52.02, each asymmetric unit containing two molecules.     

慈菇蛋白酶抑制剂的一级结构

慈菇蛋白酶抑制剂A和B经硫硫键还原及羧甲基化后,分别用内肽酶及溴化氰裂解,肽段经分离纯化后在气相蛋白质序列仪上进行序列分析,通过重叠肽段的拼接,阐明了全部一级结构.慈菇抑制剂A和B均由150个氨基酸残基组成,含有三对硫硫键.两者在结构上有91％的同源性,但与其它已知结构的丝氨酸蛋白酶抑制剂有明显不同.因而它是属于新的一族抑制剂.根据两者的结构特征.推测它们的活性中心均为Lys-Ser(44—45)和Arg-Tyr-Lys(77—79).在抑制剂A,B 13个残基差异中,其中第87位残基由Leu置换为Arg.这可能是引起两者抑制特性差异的主要原因.     

天花粉胰蛋白酶抑制剂及其类似物的全合成

天花粉胰蛋白酶抑制剂(Trichosanthes trypsin inhibitor, TTI)是一含27个氨基酸残基的多肽,其中包括三对硫硫键。本文报道了它的化学全合成及其分子内二硫键重组,合成产物纯化后其氨基酸序列以及与胰蛋白酶的抑制当量比均与天然抑制剂相一致。并合成了此抑制剂的类似物,其N端第6位Met残基由Ala所取代,合成产物纯化后,其抑制活力也与天然抑制剂相同。此结果为今后天花粉胰蛋白酶抑制剂作为融合蛋白用基因工程表达以及产物的后处理提供了依据。     

绿豆胰蛋白酶抑制剂片段及其类似物的合成

绿豆胰蛋白酶抑制剂的Lys活性碎片由两条分别含有26及9个氯基酸残基的多肽链通过两对分子间二硫键连接而成。用DTT还原能拆分两链,其中长链含6个半胱氨酸,在空气中氧化后能恢复25％原Lys碎片活力。本文报道了此长链的化学合成和二硫键重组。合成产物的氯基酸组成与文献报道的一致。但活性明显低于天然产物。为此对绿豆抑制剂的部分序列重新进行测定,结果表明原P_2′位的Lys应为Ile按新测定序列,从长链26肽的N端和C端各去掉两个残基合成一个22肽,此22肽的活性与天然的26肽相当。此外还合成了此22肽的类似物,其活性中心的Lys残基由Ala取代,产物对胰蛋白酶和弹性蛋白酶都无抑制活力。