STRUCTURE & ASSEMBLY OF COLLAGEN MOLECULES & FIBRILS

这是我们继续申请的第二次修订。我们的目标是了解胶原分子组装和原纤维形成的结构和机制。I型胶原蛋白是通常由两条α 1和一条α 2链组成的异源三聚体。I型胶原的分子组装始于C-前肽的注册。有效的前胶原I异源三聚体组装似乎需要一种正确的链识别机制必须存在于ER内。生物合成途径中的一个悬而未决的问题仍然是如何选择不同的基因产物;对齐并随后折叠成三螺旋。我们建议进行以下三项研究。(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。将融合蛋白结晶，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融合前肽也将用于使用光散射和分析超离心程序研究蛋白质-蛋白质相互作用。(2)用酵母双杂交系统测定C-前肽在体内条件下的相互作用。C-前肽的不同结构域将表达为具有Gal 4激活剂结构域的融合蛋白。将测试这些肽与表达为具有Gal 4 DNA结合结构域的融合蛋白的全长C-前肽的相互作用。(3)利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-Gal 4DNA结合区融合蛋白将用作诱饵以分离相互作用的细胞蛋白。将检查分离的蛋白质在C-前肽相互作用中的作用。最后一项研究（4）将检查与分子组装成原纤维相关的N-端肽结构和调节包装的交联模式。这些研究将有助于了解人类的功能障碍。

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.