MIMICS OF HELIX TURN HELIX PEPTIDES

螺旋转角螺旋肽的模拟物

• 批准号: 6180628
• 负责人: FELICIA A ETZKORN
• 金额: $ 4.37万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2000-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180628
• 关键词: DNA binding protein; chimeric proteins; conformation; crosslink; nuclear magnetic resonance spectroscopy; peptide analog; peptide chemical synthesis; protein engineering; protein folding; protein structure; protein structure function; stereochemistry; structural biology; transcription factor

DESCRIPTION: The goal of this project is to mimic the helix-turn-helix (HTH) motif of the POU Oct-1 homeodomain and the 434 Cro repressor by constraining the native conformation of this small portion of the protein. The helix-turn-helix motif is found in several DNA-binding proteins that have known 3-dimensional structures. Such chimeric molecules can serve a two-fold health-related purpose. First, the design and biochemical evaluation of the chimeric molecules will explore fundamental aspects of protein folding and protein-DNA interactions. Second, each of the mimics described could serve as biochemical probes of developmental processes. POU homeodomains are transcription factors for human growth hormone, histones, snRNAs, immunoglobulin, herpes simplex virus and other medically important genes. The PI proposes to constrain the conformation of peptides corresponding to the HTH motif via covalent bonds between buried hydrophobic side-chains. Two approaches to this unusual type of side-chain linkage will be taken: 1) aryl linkage and 2) alkyl linkage. The templates are designed to stabilize the conformation of the flexible turn. The designs feature stable organic side-chains which will favor protein folding by burial of the additional hydrophobic moiety. Each template will be synthesized stereospecifically from amino acid starting materials and incorporated by solid-phase synthesis into a peptide corresponding to the sequence of the structurally well-characterized Cro repressor and POU Oct-1 homeodomain. Peptide engineering of the alpha-helices will introduce salt bridges and residues with high helix propensities to improve the helicity of attached peptides. A helix-to-helix crosslink will be introduced to improve the tertiary structure stability. The HTH mimics will be characterized chemically (HPLC, MS, NMR), structurally (CD, NMR and/or x-ray crystallography) and functionally (DNA-binding affinity) to ascertain the success of the mimics.

描述：该项目的目标是模仿螺旋-转角-螺旋 (HTH)POU Oct-1同源结构域和434 Cro阻遏物的基序， 限制了这一小部分蛋白质的天然构象。 螺旋-转角-螺旋基序存在于几种DNA结合蛋白中， 具有已知的三维结构。 这样的嵌合分子可以作为一种 双重健康目的。 一、设计与生化 嵌合分子的评估将探索 蛋白质折叠和蛋白质-DNA相互作用。 第二，每一个模仿者 可以作为发育过程的生物化学探针。 POU 同源结构域是人生长激素，组蛋白， snRNA、免疫球蛋白、单纯疱疹病毒和其他医学上重要的 基因. PI建议限制肽的构象 对应于HTH基序通过共价键之间埋疏水 侧链 有两种方法来处理这种不寻常的侧链连接， 可以采用：1）芳基连接和2）烷基连接。 模板设计 以稳定柔性圈的构造。 设计特点 稳定的有机侧链，这将有利于蛋白质折叠的埋葬 另外的疏水部分。 每个模板将被合成 立体特异性地由氨基酸起始材料制备，并通过 固相合成为对应于所述多肽的序列的肽。 结构上充分表征Cro阻遏物和POU Oct-1同源结构域。 α-螺旋的肽工程将引入盐桥， 具有高螺旋倾向的残基，以改善连接的 缩氨酸 将引入螺旋-螺旋交联以改善 三级结构稳定性 HTH模拟物将被表征为 化学上（HPLC，MS，NMR），结构上（CD，NMR和/或X射线 晶体学）和功能（DNA结合亲和力），以确定 模仿者的成功