STRUCTURE & ASSEMBLY OF COLLAGEN MOLECULES & FIBRILS

这是我们的延续申请的第二次修改。我们的目标是了解胶原蛋白分子组装和纤维形成的结构和机制。I型胶原是一种异源三聚体，通常由两条al链和一条a2链组成。I型胶原的分子组装始于c -前肽的登记。有效的前胶原I异源三聚体组装似乎需要内质网中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仍然是如何选择不同的基因产物；排列并随后折叠成三螺旋结构。我们提出以下三项研究。(1)确定c -前肽的结构，并在体外检测各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，并用分子置换法确定嵌合蛋白的结构。将比较类似的C-前肽结构域的折叠。GST融合前肽也将使用光散射和分析超离心程序来研究蛋白质-蛋白质相互作用。(2)利用酵母双杂交系统确定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与Gal4激活子结构域的融合蛋白。这些将被测试与全长c -前肽的相互作用，表达为与Gal4 DNA结合域的融合蛋白。(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- gal4 DNA结合区融合蛋白将被用作分离相互作用的细胞蛋白的诱饵。分离的蛋白质将被检查它们在c -前肽相互作用中的作用。最后的研究(4)将检查与分子组装成原纤维和调节包装的交联模式相关的n端肽结构。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.