Structural Studies of Triple-helical Proteins

描述（由申请人提供）：尽管胶原蛋白在细胞外基质中具有主要的结构和信号作用，并参与许多疾病，但我们对基本的三螺旋特征以及它们如何受到致病突变的影响的理解仍存在空白。缺乏可接近的重组胶原蛋白系统限制了基于诱变的结构、生物活性位点、致病突变和药物筛选的探索。目前，还没有已知的小分子药物能与胶原蛋白结合用于临床应用。这项工作的长期目标是建立一个重组系统来生产基于胶原蛋白的组织工程支架，并发现与胶原蛋白相互作用以抑制致病过程的药物。目标1提出获得关于带电对相互作用和三螺旋弯曲的基本知识。由于高含量的带电残基及其参与胶原蛋白功能的各个层面，一种结合实验和计算的方法将定义链内和链间的贡献，并比较转置带电对。数据表明，三螺旋分子并不总是具有线性结构，最近开发的集成溶液结构方法，使用x射线散射，分析超离心和约束建模，将应用于定义三螺旋的弯曲

DESCRIPTION (provided by applicant): In spite of the major structural and signaling roles of collagen in the extracellular matrix, and its involvement in many diseases, there are gaps in our understanding of basic triple-helix features and how they are affected by pathogenic mutations. The absence of an accessible recombinant collagen system limits mutagenesis-based exploration of structure, biologically active sites, pathogenic mutations, and drug screening. At this time, no small molecule drugs are known to bind to collagen for clinical applications. The long-term goals of this work are to establish a recombinant system to produce collagen-based scaffolds for tissue engineering and to discover drugs that will interact with collagen to inhibit pathogenic processes. Aim #1 proposes to acquire fundamental knowledge about charged pair interactions and triple-helix bending. Motivated by the high content of charged residues and their involvement at every level of collagen function, a combined experimental and computational approach will define intra vs. interchain contributions and compare transposed charged pairs. Data suggest triple-helical molecules do not always have a linear structure, and a recently developed integrated solution structure approach, using x-ray scattering, analytical ultracentrifugation and constrained modeling, will be applied to define bending of the triple-helix and its sequence dependence. The objective of Aim #2 is to markedly improve an existing recombinant bacterial collagen system as a model for human collagen by introducing appropriate proline hydroxylation and by establishing the capacity to form heterotrimers using a coiled coil domain for chain selection. The structural and biological effectiveness of these enhancements will be tested by inserting within the bacterial collagen domain a human collagen platelet binding site requiring hydroxylation for platelet aggregation activity and a collagenase cleavage site requiring heterotrimers for activity. Aim #3 is directed towards elucidating the mechanism through which Gly missense mutations in collagen lead to hereditable connective tissue disorders. The misfolding of mutant collagens appears to lead to degradation, through endoplasmic reticulum (ER) stress, UPR or autophagy, while mutation interference with collagen binding to cell receptors may represent an alternate mechanism in select cases. The direct effect of Gly missense mutations on triple-helix folding and on integrin binding will be determined on a bacterial system, and the detailed structural effects defined in model peptides, providing quantitative data to clarify the disease mechanism. The proposed research is significant because it will move the field forward in terms of foundational knowledge about basic triple-helix properties and will provide tools for finding drugs that can correct collagen defects. The innovative establishment of a recombinant collagen system with the capacity for hydroxylation of proline and heterotrimer formation creates a substantive new capacity to model and modify biologically important sites from human collagens and is well suited for initial screening of small molecules that can accelerate collagen folding, promote receptor binding, or inhibit degradation.