DESIGN OF SEQUENSE-SELECTIVE DNA-BINDING SMALL PROTEINS

我们采用基于结构的设计，从f -肌动蛋白捆绑蛋白绒毛蛋白中构建了一个小的折叠结构域，该结构域含有碱性亮氨酸拉链蛋白GCN4 DNA结合所需的氨基酸，并将其与GCN4的DNA结合进行了比较。单体基序折叠成稳定结构域，以刚体机制与DNA结合，但其亲和力不高于碱性区域肽。将亮氨酸拉链区添加到折叠结构域恢复了其序列特异性DNA结合，与GCN4相当。与单体折叠结构域不同，其亮氨酸拉链衍生物在DNA结合时发生构象变化。CD光谱和热力学研究表明，在DNA存在或不存在的情况下，DNA接触区是折叠的，并且表明在DNA存在的情况下，DNA接触区和亮氨酸拉链区之间的连接转变为螺旋结构。这些结果表明，直接接触区外的结构转变调节了dna接触区的精确位置，在碱性亮氨酸拉链蛋白的特异性复合物形成中起着关键作用。另一种设计策略是利用折叠良好的C2H2锌指基序小结构域作为DNA结合α-螺旋的支架。将C2H2锌指基序的α-螺旋部分适当替换为GCN4序列选择性结合所必需的氨基酸残基，将提供具有折叠结构的新型DNA结合域。目前正在进行结构方面的研究以及在各种金属离子存在下新蛋白的序列特异性DNA结合。

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4, and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain, and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. Addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon the DNA binding. CD spectral and thermodynamic studies indicate that the DNA -contacting region is folded in the presence or absence of DNA, and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of the basic leucine zipper proteins.Another design strategy utilized a well-folded small domain of C2H2 zinc finger motif as scaffold for the DNA binding α-helix. Appropriate substitution of the α-helical portion of the C2H2 zinc finger motif with amino acid residues necessary for the sequence-selective binding of GCN4 would afford a novel DNA binding domain with a folded structure. Studies on struacural aspects and the sequence-specific DNA binding of the novel protein in the presence of various metal ions are currently underway.