MOLECULAR CHARACTERIZATION OF SUGAR TRANSPORT CARRIER

糖运输载体的分子表征

• 批准号: 3225032
• 负责人: CHAN Y. JUNG
• 金额: $ 7.94万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225032
• 关键词: X ray crystallography; affinity labeling; chemical binding; chemical structure function; circular dichroism; conformation; crosslink; erythrocytes; fluorescence spectrometry; glucose transport; infrared spectrometry; membrane proteins; protein sequence; transport proteins

This proposal is a cont1nuing effort to elucidate the molecular mechanism of the human erythrocyte glucose transporter function. The long term objects are to understand the nature of the defect underlying insulin-resistive states seen in Type II diabetes and obesity. More immediate objectives are to determine tertiary structure of the transporter and the conformational dynamics associated with the transport function. The amino acid sequence of this protein is known, which predicts that the protein is made of twelve transmembrane segments and four nontransmembrane hydrophilic segments. Based on these predictions, the following experiments are proposed. First, these transmembrane and nontransmembrane segments will be isolated and identified by protease-digestion. HPLC-separation and determination of characterlstic amino acid contents predicted by the model. The three dimensional arrangement of these segments will be determined by labelling them with crosslinking reagents, lipophilic covalent probes, and tritiated water protons. Possible changes in these three dimensional arrangements upon the removal of the nontransmembrane, hydrophilic segments will be monitored by the same procedures. The changes will also be monitored based on circula, dichroism, Fourier transform infrared spectroscopy, radiation inactivation target size and fluorescence energy transfer measurements. The substrate binding sites in the substrate recognition pockets and in the translocatlon pathway in the protein will be studied by tagging them with photoreactive substrate analogs. Side-specific, nontransportable analogs and transportable analogs will be used to differentiate these sites. Cytochalasin B binding pocket will be characterized by identifying its binding site. Those transmembrane or nontransmembrane hydrophilic segments that are affected by the substrates or inhibitor-induced conformational perturbation will be determined by labelling and identifying the amino acid residues whose reaction to certain alkylating reagents are known to inactivate the protein function and are sensitive to the substrate and inhibitors. Four different alkylating reagents will be used for this purpose. Lastly, as a long term goal, crystallization of the purified transporter protein will be attempted for future x-ray diffraction studies.

这一建议是一个持续的努力，以阐明分子 人红细胞葡萄糖转运蛋白功能的机制。 长期目标是了解缺陷的性质 在II型糖尿病中观察到的潜在胰岛素抵抗状态， 肥胖 更直接的目标是确定高等教育 转运蛋白的结构和构象动力学 与运输功能有关。 氨基酸序列 这种蛋白质是已知的，这预示着蛋白质是由 十二个跨膜片段和四个非跨膜片段 亲水链段。 根据这些预测， 实验提出。 首先，这些跨膜和 非跨膜片段将被分离和鉴定， 蛋白酶消化 高效液相色谱法分离和测定 用该模型预测的特征氨基酸含量。 的 将确定这些段的三维布置 通过用交联剂、亲脂性共价键 探针和氚化水质子。 这些可能的变化 三维布置， 非跨膜亲水片段将由 同样的程序。 这些变化也将根据 圆图，二向色性，傅里叶变换红外光谱， 辐射灭活靶大小和荧光能量转移 测量. 底物中的底物结合位点 识别口袋和蛋白质中的移位途径 将用光敏底物标记它们， 类似物 侧特异性、非转运类似物和可转运类似物 类似物将用于区分这些位点。 松弛素 B结合口袋将通过鉴定其结合来表征 绝佳的价钱 那些跨膜或非跨膜亲水片段 受底物或底物诱导的 构象扰动将通过标记来确定， 确定氨基酸残基，其与某些 已知烷基化试剂会破坏蛋白质功能 并且对底物和抑制剂敏感。 四个不同 烷基化试剂将用于此目的。 最后，作为 长期目标，纯化的转运蛋白的结晶 将尝试用于未来的X射线衍射研究。