NMR Studies of Triple helical Peptides

三螺旋肽的核磁共振研究

• 批准号: 7933138
• 负责人: JEAN S BAUM
• 金额: $ 27.04万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933138
• 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）：赠款的广泛长期目标是使用NMR来洞悉氨基酸序列中的变化如何影响构象，动力学和胶原蛋白（Gly-X-Y）N Triple helix基序的折叠。这些研究将用于了解胶原蛋白的生物学意义，特别是与识别和疾病有关的胶原蛋白。具体目的1：在纤维状胶原三螺旋蛋白的三螺旋中，基质金属蛋白酶（MMP）裂解位点的两个定义明确的相互作用位点，对结缔组织的正常降解至关重要，A2B1整联蛋白结合位点的正常降解，对局部综合性和背面的模式进行了研究，以确定局部综合性的模型，以确定局部综合剂的动态，并将其反向研究。相邻残基在调节识别位点的作用。特定的目标＃2：在Gly-X-Y重复模式中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。 NMR研究将阐明中断的性质，并检验这些断裂改变结构并用作识别位点的假设。特定目的＃3：另一个氨基酸替代单个gly会破坏特征重复（GLY-X-Y）N序列模式，并导致结缔组织疾病可能对非致命表型具有致命性。从gly到X突变引起的变化可能会发生在不同级别的不同级别，从三螺旋的折叠到损失高阶功能，例如配体结合或自识别。 NMR构象，动力学和模型肽的折叠研究将检验以下假设：GLY突变通过改变主链波动来减少临界识别位点的结合亲和力。特定目的＃4：现代NMR技术，包括测量二面角和距离约束，将通过使用偶有标记的肽通过肽合成或通过重组细菌表达系统获得的偶有标记的肽来适应三重螺旋。了解结构/功能相关性以及酶认识到三重螺旋的机制可能会提供信息，这些信息将有助于开发靶标，以治疗多种疾病，包括骨关节炎和癌症，并有助于发现胶原蛋白疾病的新疗法。