STRUCTURE & ASSEMBLY OF COLLAGEN MOLECULES & FIBRILS

结构

• 批准号: 6452651
• 负责人: ARTHUR VEIS
• 金额: $ 30.42万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-03-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6452651
• 关键词: X ray crystallography; affinity chromatography; binding proteins; collagen; conformation; connective tissue metabolism; extracellular matrix; fibrogenesis; intermolecular interaction; laboratory rabbit; laboratory rat; peptide structure; procollagen; protein biosynthesis; protein protein interaction; protein sequence; protein structure function; ultracentrifugation; western blottings; yeast two hybrid system

This is the second revision of our continuation application. Our goal has been to understand the structure and mechanism of collagen molecule assembly and fibril formation. Type I collagen is a heterotrimer normally composed of two al and one a2 chains. Molecular assembly of type I collagen begins with the registration of the C-propeptides. Efficient procollagen I heterotrimer assembly appears to require that a mechanism for correct chain recognition must exist within the ER. An open question in the biosynthetic pathway remains as to how the different gene products are selected; aligned and subsequently folded into the triple helix. We propose the following three studies. (1) Determine the structures of the C-propeptides and examine in vitro the interactions between the various domains. The different domains of the C-propeptides will be synthesized as GST fusion proteins. The fusion proteins will be crystallized and the chimeric protein structures will be determined using the molecular replacement method. The folding of the comparable C- propeptide domains will be compared. The GST fusion propeptides will also be used to study protein-protein interactions using light-scattering and analytical ultra-centrifugation procedures. (2) Determine the interactions of the C-propeptides under in vivo conditions using the yeast two-hybrid system. The different domains of the C-propeptides will be expressed as fusion proteins with the Gal4 activator domain. These will be tested for interaction with the full length C-propeptides expressed as fusion proteins with the Gal4 DNA binding domain. (3) Identify the cell proteins that bind to C-pro a1 and C-pro a2 using affinity chromatography and the yeast two-hybrid system. The C- propeptide-Gal4 DNA binding region fusion protein will be used as a bait to isolate interacting cell proteins. The proteins isolated will be examined for their role in the C-propeptide interactions. The final study (4) will examine N-telopeptide structures as related to molecular assembly into fibrils and the cross-link patterns that modulate packing. These studies will help in understanding dysfunction in humans.

这是我们的延续申请的第二次修订。我们的目标是了解胶原蛋白分子组装和原纤维形成的结构和机制。 I型胶原蛋白是异源三聚体，通常由两条α1链和一条α2链组成。 I 型胶原蛋白的分子组装始于 C 前肽的注册。有效的 I 型前胶原异源三聚体组装似乎要求 ER 内必须存在正确的链识别机制。生物合成途径中的一个悬而未决的问题是如何选择不同的基因产物。对齐并随后折叠成三螺旋。我们提出以下三项研究。 (1)确定C-前肽的结构并在体外检查各个结构域之间的相互作用。 C 前肽的不同结构域将被合成为 GST 融合蛋白。融合蛋白将被结晶，并使用分子置换方法确定嵌合蛋白结构。将比较可比较的C-前肽结构域的折叠。 GST 融合前肽还将用于通过光散射和分析超速离心程序研究蛋白质-蛋白质相互作用。 (2) 使用酵母双杂交系统确定体内条件下C-前肽的相互作用。 C 前肽的不同结构域将表达为与 Gal4 激活剂结构域的融合蛋白。将测试它们与表达为具有 Gal4 DNA 结合域的融合蛋白的全长 C 前肽的相互作用。 (3) 使用亲和层析和酵母双杂交系统鉴定与 C-pro a1 和 C-pro a2 结合的细胞蛋白。 C-前肽-Gal4 DNA 结合区融合蛋白将用作分离相互作用细胞蛋白的诱饵。将检查分离的蛋白质在 C 前肽相互作用中的作用。最终研究 (4) 将检查与分子组装成原纤维相关的 N-端肽结构以及调节堆积的交联模式。这些研究将有助于了解人类功能障碍。