STRUCTURAL STUDIES OF CONNECTIVE TISSUES

基底膜固有的独特成分 特征，但IV型胶原蛋白的三维组织和 它与其他组成部分结构关系仍有待确定。 x射线 干燥、拉伸的牛透镜囊的衍射图案显示， 除了IV型三螺旋的高角度反射外， 4.6nm和33.5nm的单色反射，这表明了令人惊讶的程度 超分子的秩序。 我们建议澄清x之间的关系- 射线数据和天然基底膜结构。 的起源 将识别在特定组织成分中观察到的重复单元 通过对选择性提取的透镜囊和 单个组件。 电子显微镜检查将在拉伸和 未拉伸的透镜囊，通过在冷冻蚀刻上复制电镀制备 样品，和光学衍射的电子显微镜照片将进行比较 X射线衍射图。 透镜囊的结构信息， 它代表了一个高度有序和IV型丰富的基底膜， 为理解其他组织的结构和功能提供基础 基底膜和疾病状态的改变。 解释我们的基底膜x射线数据需要一个模型， IV型胶原的分子构象。 经典的三螺旋 在纤维状胶原中发现的构象具有严格的氨基酸 限制是每三个残基必须被甘氨酸和Gly取代 与结缔组织疾病有关。 相比之下， IV型胶原蛋白在重复三肽中含有超过20个断裂 （X-Y-Gly）n模式。 我们假设：（1）中断的影响 取决于其一般特征[Gly取代、Gly缺失、Y（或 X）缺失或插入];和（2）围绕一个或多个X）缺失或插入的带电对序列。 中断通过允许肽 链在现场循环。 这些假设将由以下人员进行检验： 表征构象、稳定性和折叠限定序列 三螺旋肽，包括具有侧翼为 带电三重态，使用圆二色性和NMR光谱，一起 用X射线衍射和计算机建模。 关于简单的研究 模型，具有34个残基的IV型序列的肽，其包括 将对中断进行表征，并将使用二维核磁共振来 确定“设计的”三螺旋的三维构象 溶液中的肽。 这些肽研究的结果将有助于确定 第四型三螺旋的扰动，并澄清结构 纤维胶原突变的后果。

The unique components intrinsic to basement membranes have been characterized, but the 3-dimensional organization of type IV collagen and its structural relation to other components remain to be defined. X-ray diffraction patterns of dried, stretched bovine lens capsule show, in addition to high angle reflections from the type IV triple-helix, series of 4.6nm and 33.5 nm meridional reflections which suggest a surprising degree of supramolecular order. We propose to clarify the relation between the x- ray data and native basement membrane structure. The origin of the observed repeating units in specific tissue components will be identified through x-ray studies on selectively extracted lens capsules and on individual components. Electron microscopy will be done on stretched and unstretched lens capsules, prepared by replica plating on freeze etched specimens, and optical diffraction of electron micrographs will be compared with x-ray diffraction patterns. Structural information on lens capsule, which represents a highly ordered and type IV rich basement membrane, will provide a basis for understanding the structure and function of other basement membranes and alterations in diseased states. Interpretation of our basement membrane x-ray data requires a model for the molecular conformation of type IV collagen. The classic triple-helical conformation found in fibrillar collagens has the stringent amino acid constraint that every third residue must by glycine, and Gly substitution have been associated with connective tissue diseases. In contrast, native type IV collagen contains more than 20 breaks in the repeating tripeptide (X-Y-Gly)n pattern. We hypothesize: (1) the effect of an interruption depends upon its general character [Gly substitution, Gly deletion, Y (or X) deletion, or insertion]; and (2) charged pair sequences surrounding an interruption lessen the structural consequences by allowing the peptide chain to loop out at the site. These hypotheses will be tested by characterizing the conformation, stability and folding defined sequence triple-helical peptides, including peptides with interruptions flanked by charged triplets, using circular dichroism and NMR spectroscopy, together with x-ray diffraction and computer modeling. Following studies on simple models, a peptide with a 34 residue type IV sequence which includes an interruption will be characterized, and 2-dimensional NMR will be used to determine the 3-dimensional conformation of a "designed" triple-helical peptide in solution. Results from these peptide studies will help define perturbations to the type IV triple-helix, and clarify the structural consequences of fibrillar collagen mutations.