Sieber M, Allemann R K
Laboratory for Organic Chemistry, Department of Chemistry, ETH-Zürich, Universitätstrasse 16, CH-8092 Zurich, Switzerland.
Nucleic Acids Res. 1998 Mar 15;26(6):1408-13. doi: 10.1093/nar/26.6.1408.
By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.
通过设计含有MASH-1的BHLH结构域编码区串联拷贝（MASH-BHLH）的重组基因，并带有编码8个或17个氨基酸连接序列的间隔DNA序列，MASH二聚体的两个亚基已被连接形成单链二聚体MM8和MM17。尽管连接第一个BHLH亚基C末端与第二个亚基N末端的连接子长且灵活，距离约为55埃，但单链二聚体能够在大肠杆菌中高水平产生。MM8和MM17是单体，未观察到亚基的“交叉折叠”。圆二色光谱显示，与野生型MASH-BHLH一样，MM8和MM17在不存在DNA时仅采用部分折叠结构，但在结合DNA时会发生折叠转变，形成主要为α-螺旋的构象。通过电泳迁移率变动分析进行的滴定显示，与野生型MASH-BHLH相比，单链二聚体对含E盒DNA序列的亲和力增加了约10倍。另一方面，对异源DNA序列的亲和力仅增加了5倍。因此，肽连接子的引入导致DNA结合特异性从-0.14千卡/摩尔增加到-0.57千卡/摩尔，增加了4倍。
