NMR Studies of Triple helical Peptides

三螺旋肽的核磁共振研究

• 批准号: 7626020
• 负责人: JEAN S BAUM
• 金额: $ 26.18万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7626020
• 关键词: Affect; Affinity; Amino Acid Sequence; Amino Acids; Arthritis; Basement membrane; Binding; Binding Sites; C-terminal; Cell Adhesion Process; Cells; Characteristics; Collagen; Collagen Diseases; Connective Tissue; Connective Tissue Diseases; Degenerative polyarthritis; Development; Disease; Enzymes; Extracellular Matrix; Fibrillar Collagen; Gene Mutation; Grant; Hydroxyproline; Individual; Integrin Binding; Interruption; Investigation; Label; Ligand Binding; Ligands; Malignant Neoplasms; Matrix Metalloproteinases; Measurement; Methods; Modeling; Molecular; Molecular Conformation; Mutation; N-glycylalanine; Nature; Neoplasm Metastasis; Non-Fibrillar Collagens; Pattern; Peptide Synthesis; Peptides; Phenotype; Process; Proteins; Protocols documentation; Recombinants; Research Personnel; Residual state; Rheumatologic Disorder; Role; Severity of illness; Shapes; Site; Solutions; Structural Protein; Structure; System; Techniques; Testing; Thrombosis; Variant; Vertebral column; base; collagenase; design; flexibility; insight; mutant; novel; programs; research study; restraint; synthetic peptide; triple helix; tumor

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.

描述（由申请人提供）：该资助的广泛长期目标是使用NMR来深入了解氨基酸序列的变化如何影响胶原蛋白（Gly-X-Y）n三螺旋基序的构象、动力学和折叠。这些研究将用于了解胶原蛋白中的生物学重要位点，特别是与识别和疾病相关的位点。具体目标1：在纤维状胶原的三螺旋中的两个明确的相互作用位点，基质金属蛋白酶（MMP）切割位点，对结缔组织的正常降解至关重要，和α 2 β 1整联蛋白结合位点，对细胞粘附过程至关重要，将通过NMR进行检查，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位点。具体目标#2：Gly-X-Y重复模式中的断裂通常在基底膜中的非纤维状胶原的三螺旋结构域中发现。NMR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：单个Gly被另一个氨基酸取代打破了特征性重复（Gly-X-Y）n序列模式，并导致可能具有致死至非致死表型的结缔组织疾病。由Gly至X突变引起的改变可能发生在不同的水平上，从三螺旋的折叠缺陷到高级功能的丧失，例如配体结合或自我识别。NMR构象，动力学和折叠模型肽的研究将测试的假设，即甘氨酸突变降低关键识别位点的结合亲和力，通过改变骨干波动。具体目标#4：包括二面角和距离限制的测量的现代NMR技术将通过使用双标记肽来适应三螺旋，所述双标记肽将通过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以提供有助于开发治疗多种疾病（包括骨关节炎和癌症）的靶点的信息，并有助于发现胶原蛋白疾病的新疗法。