NMR Studies of Triple helical Peptides

描述(由申请人提供)：该基金广泛的长期目标是使用核磁共振来深入了解氨基酸序列的变化如何影响胶原(Gly-X-Y)n三螺旋基序的构象、动力学和折叠。这些研究将被应用于了解胶原蛋白中具有生物学意义的部位，特别是那些与识别和疾病有关的部位。具体目标#1：核磁共振将研究纤维胶原蛋白三螺旋中的两个明确的相互作用部位，对结缔组织的正常降解至关重要的基质金属蛋白酶(MMP)裂解部位，以及对细胞黏附过程至关重要的a2b1整合素结合部位，以确定局部构象和骨架动力学，以阐明不同的识别模式，并了解相邻残基在调节识别部位中的作用。特定目的#2：Gly-X-Y重复模式的断裂通常在基底膜的非纤维胶原蛋白的三螺旋结构域中发现。核磁共振研究将阐明中断的性质，并测试这些中断改变结构并作为识别位点的假设。具体目标#3：用另一种氨基酸取代单个甘氨酸，打破了(Gly-X-Y)n重复序列模式，导致结缔组织疾病，这种疾病可能具有致命性或非致命性表型。由Gly到X突变引起的改变可能发生在不同的水平上，从三螺旋的缺陷折叠到高阶功能的丧失，如配体结合或自我识别。对模型多肽的核磁共振构象、动力学和折叠研究将检验这一假设，即Gly突变通过改变骨架波动来降低关键识别位点的结合亲和力。具体目标#4：现代核磁共振技术，包括测量二面角和距离限制，将通过使用双标记多肽来适应三螺旋结构，这些双标记多肽将通过多肽合成或通过重组细菌表达系统获得。了解结构/功能相关性和酶识别三螺旋的机制可能会提供有助于开发治疗包括骨关节炎和癌症在内的几种疾病的靶点的信息，并将有助于发现胶原蛋白疾病的新疗法。

DESCRIPTION (provided by applicant): The broad long term objectives of the grant are to use NMR to provide insight into how variations in amino acid sequence affect conformation, dynamics, and folding of the collagen (Gly-X-Y)n triple helix motif. These studies will be applied to understand biologically significant sites in collagen, in particular those related to recognition and disease. Specific Aim #1: Two well-defined interactions sites in the triple helix of fibrillar collagens, the matrix metalloproteinase (MMP) cleavage site, critical to normal degradation of connective tissue, and the a2b1 integrin binding site, critical to cell adhesion processes, will be examined by NMR to determine local conformation and backbone dynamics to elucidate the different modes of recognition and to understand the role of neighboring residues in modulating recognition sites. Specific Aim #2: Breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domain of non fibrillar collagens in basement membrane. NMR investigations will elucidate the nature of the interruptions and test the hypothesis that these breaks alter structure and serve as recognition sites. Specific Aim #3: The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)n sequence pattern and results in connective tissue disease that can have lethal to non-lethal phenotypes. Alterations arising from Gly to X mutations may occur at different levels ranging from defective folding of the triple-helix to loss of higher order function such as ligand binding or self recognition. NMR conformation, dynamics and folding studies on model peptides will test the hypothesis that Gly mutations reduce the binding affinities in critical recognition sites by altering the backbone fluctuations. Specific Aim #4: Modern NMR techniques including the measurement of dihedral angles and distance restraints will be adapted to the triple helix by use of doubly labeled peptides that will be obtained either by peptide synthesis or through a recombinant bacterial expression system. Understanding structure/function correlation and the mechanism by which enzymes recognize the triple helix may provide information that will aid in in the development of targets for the treatment of several diseases including osteoarthritis and cancer, and will aid in the discovery of new therapies for collagen diseases.