Krylov D, Olive M, Vinson C
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
EMBO J. 1995 Nov 1;14(21):5329-37. doi: 10.1002/j.1460-2075.1995.tb00217.x.
We appended a rationally designed acidic amphipathic protein sequence to the N-terminus of a leucine zipper. Circular dichroism data indicate that this engineered polypeptide sequence can 'zipper' up the basic region of a bZIP monomer into a heterodimeric coiled coil. This propagation of the leucine zipper dimerization interface into the basic region can proceed for up to four heptads and stabilizes the heterodimer complex 2.5 kcal/mol or > 100-fold. The acidic nature of the extension is the most critical component of the design, suggesting that the extension is acting as a DNA mimetic. The dimerization prevents the basic region in this heterodimeric coiled coil structure from binding to DNA. Gel-shift, fluorescence and transient transfection assays indicate that the acidic extension appended to a leucine zipper can inactivate the DNA-binding and transactivation properties of the bZIP protein C/EBP. The three bZIP basic regions examined in this study dimerize with similar stability with the acidic extension, suggesting that this N-terminal extension can be used to develop dominant-negatives to other bZIP transcription factors.
我们在亮氨酸拉链的N端附加了一个经过合理设计的酸性两亲性蛋白质序列。圆二色性数据表明，这种工程化的多肽序列能够将bZIP单体的碱性区域“拉链”成异源二聚体卷曲螺旋。亮氨酸拉链二聚化界面向碱性区域的这种延伸最多可进行四个七肽重复，并使异源二聚体复合物稳定2.5千卡/摩尔或>100倍。延伸部分的酸性性质是设计中最关键的组成部分，这表明该延伸部分起着DNA模拟物的作用。二聚化阻止了这种异源二聚体卷曲螺旋结构中的碱性区域与DNA结合。凝胶迁移、荧光和瞬时转染实验表明，附加到亮氨酸拉链上的酸性延伸部分能够使bZIP蛋白C/EBP的DNA结合和反式激活特性失活。本研究中检测的三个bZIP碱性区域与酸性延伸部分以相似的稳定性二聚化，这表明该N端延伸部分可用于开发针对其他bZIP转录因子的显性负性突变体。
