大肠杆菌二氢硫辛酸转乙酰基酶的外周亚基结合结构域的溶液构象

Soluble Conformation of Peripheral Subunit-binding Domain of E. coli Dihydrolipoyl Transacetylase

丙酮酸脱氢酶复合体催化丙酮酸氧化脱羧,生成乙酰辅酶A.该复合体由丙酮酸脱羧酶(E1)、二氢硫辛酸乙酰转移酶(E2)和二氢硫辛酸脱氢酶(E3)三种酶组成.大肠杆菌E2的外周亚基结合结构域(peripheral subunit-binding domain,PSBD)结合E1和E3,对丙酮酸脱氢酶复合物的结构和功能有重要作用.本研究采用PCR技术扩增了E2的PSBD的48个氨基酸残基区域编码序列(c DNA),构建p ET-32a-pp-Psbd表达载体,测序正确后转入BL21(DE3)中表达,目的蛋白质用镍柱和Hi Trap SP柱纯化后达到电泳纯,质谱鉴定纯化后蛋白质分子量与理论值符合.pull-down结果表明,PSBD可分别与E1和E3结合.圆二色谱表征PSBD的二级结构主要为a-螺旋,当在0.5mol/L Na Cl的离子强度下,55.7%的PSBD分子折叠为正确的构象.动态光散射实验发现,PSBD分子有3种不同的构象存在形式,因此,PSBD非常容易从折叠态转化为不和E1、E3结合的无规卷曲态,这种构象的相互转化为其功能性与E1、E3结合及解离提供了结构基础.

The pyruvate dehydrogenase complex（ PDHc） catalyzes the oxidative decarboxylation of pyruvate and produces acyl-coenzyme A. A PDHc molecule is composed of pyruvate decarboxylase（E1）,dihydrolipoyl transacetylase（ E2） and dihydrolipoyl dehydrogenase（ E3）. The peripheral subunit-binding domain（ PSBD） of E2 from E. coli links E1 and E3,and plays an important role for PDHc assembling and function. We amplified the coding sequence（ c DNA） of the 48 amino acid residues of PSBD region using PCR,and inserted it into a p ET-32a-pp vector. The generated p ET-32app-Psbd plasmid was transfected into E. coli BL21（ DE3） for expression. The produced protein was purified by Ni-NTA and Hi Trap SP chromatography,and reached electrophoresis-grade purity. The pulldown assay results indicated that PSBD was able to bind E1 and E3. Circular dichroism（ CD） showed that the secondary structure of PSBD was mainly a-helix with 55. 7 % correctly folded PSBD in the ionic strength of 0. 5 mol / L Na Cl. Dynamic light scattering assay demonstrated that PSBD existed as three different conformations in solutions. The PSBD was easy to convert into random coil conformation andseparate from E1 and E3. Our results provided structural information for understanding the dissociation of PSBD from E1 or E3.