STRUCTURAL STUDIES OF CONNECTIVE TISSUES

基底膜固有的独特成分是 特征，但IV型胶原的三维组织和 它与其他组件的结构关系仍有待确定。X射线 干燥、拉伸的牛晶状体胶囊的衍射图显示，在 除了IV型三螺旋的高角度反射外，系列 4.6 nm和33.5 nm的子午线反射，暗示着令人惊讶的程度 超分子秩序的。我们建议澄清x-之间的关系 射线数据和天然基底膜结构。中国古代文化的起源 在特定组织成分中观察到的重复单位将被识别 通过X射线研究选择性摘除晶状体囊膜等 单个组件。电子显微镜将在拉伸和拉伸后进行 无拉伸镜片胶囊，由冷冻蚀刻复制电镀制备 将样品与光学衍射仪的电子显微照片进行比较 具有X射线衍射图。关于晶状体胶囊的结构信息， 它代表着一种高度有序和富含IV型基底膜，将 为理解其他组件的结构和功能提供基础 疾病状态下的基底膜和改变。 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 限制每三个残基必须由甘氨酸和甘氨酸取代 与结缔组织疾病有关。相比之下，原生的 IV型胶原在重复的三肽中包含20多个断裂 (X-Y-Gly)n模式。我们假设：(1)中断的影响 取决于其共性[Gly替换、Gly缺失、Y(或 X)缺失或插入]；以及(2)围绕 中断通过允许多肽减少结构后果 链条在现场循环。这些假设将通过以下方式进行验证 构象、稳定性和折叠定义序列的特征 三螺旋多肽，包括侧边带有中断的多肽 结合圆二色谱和核磁共振光谱的带电三重态 用X射线衍射和计算机模拟。以下是对SIMPLE的研究 模型，具有34个残基的IV型序列的多肽，包括一个 中断将被表征，二维核磁共振将被用于 确定“设计”的三螺旋的三维构象 溶液中的多肽。这些多肽研究的结果将有助于确定 对IV型三螺旋的扰动，并澄清了结构 纤维状胶原蛋白突变的后果。

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.