MIMICS OF HELIX TURN HELIX PEPTIDES

螺旋转角螺旋肽的模拟物

• 批准号: 6436988
• 负责人: FELICIA A ETZKORN
• 金额: $ 3.46万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 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 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6436988
• 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：这个项目的目标是模拟螺旋-旋转-螺旋 Pou Oct-1同源结构域和434 Cro阻遏物的(HTH)基序 限制了这一小部分蛋白质的天然构象。 螺旋-转弯-螺旋基序在几种DNA结合蛋白中发现 已经知道了三维结构。这种嵌合分子可以服务于 与健康相关的双重目的。第一，设计和生化 对嵌合分子的评估将探索以下基本方面 蛋白质折叠和蛋白质-DNA相互作用。其次，每一个模仿者 所描述的可作为发育过程的生化探针。POU 同源结构域是人类生长激素、组蛋白、 单链RNA、免疫球蛋白、单纯疱疹病毒和其他医学上重要的物质 基因。PI建议限制多肽的构象 通过埋藏疏水之间的共价键对应HTH基序 侧链。对这种不寻常类型的侧链连接的两种方法将 可采取：1)芳基键和2)烷基键。设计了模板 以稳定柔性转弯的构型。设计特点 稳定的有机侧链，有利于蛋白质的折叠 额外的疏水部分。每个模板都将被合成 从氨基酸起始材料立体地专一性地并通过 固相合成成与该序列对应的多肽 结构特征良好的Cro抑制子和POUOCT-1同源结构域。 α-螺旋的多肽工程将引入盐桥和 具有高螺旋倾向的残基可提高所连接的分子的螺旋度 多肽。将引入螺旋到螺旋的交联剂，以改善 三级结构稳定性。HTH的模拟物将被描述为 化学上(高效液相色谱，MS，核磁共振)，结构上(CD，核磁共振和/或X射线 结晶学)和功能(DNA结合亲和力)来确定 模仿的成功。