权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

DESIGN OF SEQUENSE-SELECTIVE DNA-BINDING SMALL PROTEINS

序列选择性 DNA 结合小蛋白的设计

基本信息

批准号：
12680590
负责人：
MORII Takashi
金额：
$ 2.56万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680590/
关键词：
DNA binding DNA RECOGNITION MOLECULAR RECOGNITION ALPHA HELIX DIMERIZATION STRUCTURE-BASED DESIGN OF PROTEIN METAL ION FOLDING タンパク質デザイン高次構造ロイシンジッパー

项目摘要

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.

我们采用基于结构的设计，从F-肌动蛋白捆绑蛋白绒毛中构建了一个小折叠结构域，该结构域包含碱性亮氨酸拉链蛋白GCN 4的DNA结合所需的氨基酸，并将其DNA结合与GCN 4进行了比较。单体基序折叠成稳定的结构域，并以刚体机制与DNA结合，而其亲和力不高于碱性区肽的亲和力。添加亮氨酸拉链区域的折叠结构域恢复其序列特异性DNA结合的GCN 4。与单体折叠结构域不同，其亮氨酸拉链衍生物在与DNA结合时发生构象变化。CD光谱和热力学研究表明，在存在或不存在DNA的情况下，DNA接触区域被折叠，并表明在存在DNA的情况下，DNA接触区域和亮氨酸拉链区域之间的连接转变为螺旋。这些结果表明，碱性亮氨酸拉链蛋白直接接触区以外的结构转变对DNA接触区的精确定位起着关键作用。另一种设计策略是利用折叠良好的C2 H2锌指结构域作为DNA结合α-螺旋的支架。将C2 H2锌指基序的α-螺旋部分适当替换为GCN 4序列选择性结合所需的氨基酸残基，将提供具有折叠结构的新DNA结合结构域。目前正在进行结构方面的研究和在各种金属离子存在下的新蛋白质的序列特异性DNA结合。