Three-dimensional Structures Of Biological Macromolecule

生物大分子三维结构

• 批准号: 6541676
• 负责人: JAMES A FERRETTI
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541676
• 关键词: DNA binding protein; Drosophilidae; arthropod genetics; binding sites; genetically modified animals; homeobox genes; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; peptide structure; protein binding; protein sequence; site directed mutagenesis; structural biology; thermodynamics

The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class, specifically the parent NK-2 homeodomain, and the full length NKX-2.5, NKx-2.8 and NKX-3.1 homeodomain containing proteins, both in the free state and bound to an uncommon 18 base-pair DNA consensus segment are being investigated. In addition, studies of the protein-protein complex made up of the NKX-2.5 and GATA-4 proteins have been initiated. Studies on mutant NKX-2.5 homeodomains associated with atrial septum defect show that either the tertiary structures of the mutant proteins are altered or their binding to the functional DNA binding sites are altered. For example, replacement of an invariant asparagine in position 51 of the homeodomain by lysine reduces the binding affinity by a factor of 1000 and alters the specificity of the protein-DNA interaction. Preliminary results suggest that this mutant results in a significant alteration of the preferred target DNA. Structural studies demonstrate that the modified homeodomain maintains the characteristic three-dimensional structure seen for most homeodomains. Thermodynamic measurements on the same wild type and mutant homeodomains are in progress. The three-dimensional structure of the NK-2 homeodomain bound to DNA, where the tyrosine in position 54 is replaced by a methionine, shows that the methionine with its longer side-chain also alters the specificity of the homeodomain-DNA interaction. The structure of another modified NK-2 homeodomain bound to DNA, where alanine in position 35 is replaced by threonine, results in a significantly altered protein structure. This structural alteration constitutes the molecular basis of early embryonic lethality associated with the mutation in the corresponding gene. Transgenic studies on the tyrosine to methionine modified homeodomain are nearing completion.

NK-2类同源结构域的野生型和突变体的三维结构，特别是亲本NK-2同源结构域，以及包含NKX-2.5、NKx-2.8和NKX-3.1全长同源结构域的蛋白质，都处于游离状态，并结合到一个罕见的18碱基对DNA共识片段，正在研究中。此外，对由NKX-2.5和GATA-4蛋白组成的蛋白-蛋白复合物的研究已经开始。对与房间隔缺损相关的突变体NKX-2.5同源结构域的研究表明，突变蛋白的三级结构被改变，或者它们与功能性DNA结合位点的结合被改变。例如，赖氨酸取代同源结构域第51位不变的天冬酰胺，将结合亲和力降低了1000倍，并改变了蛋白质- dna相互作用的特异性。初步结果表明，这种突变导致首选靶DNA的显著改变。结构研究表明，改进后的同域保持了大多数同域的特征三维结构。对同一野生型和突变型同源结构域的热力学测量正在进行中。NK-2同质结构域与DNA结合的三维结构，其中54位的酪氨酸被蛋氨酸取代，表明具有较长侧链的蛋氨酸也改变了同质结构域-DNA相互作用的特异性。另一个与DNA结合的修饰NK-2同源结构域的结构，其中35位的丙氨酸被苏氨酸取代，导致蛋白质结构发生显著改变。这种结构改变构成了与相应基因突变相关的早期胚胎致死的分子基础。酪氨酸到蛋氨酸修饰的同源结构域的转基因研究已接近完成。