MOLECULAR CHARACTERIZATION OF SUGAR TRANSPORT CARRIER

糖运输载体的分子表征

• 批准号: 3225027
• 负责人: CHAN Y. JUNG
• 金额: $ 10.74万
• 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 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225027
• 关键词: X ray crystallography; affinity labeling; chemical binding; chemical structure function; circular dichroism; conformation; crosslink; erythrocytes; fluorescence spectrometry; glucose transport; infrared spectrometry

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.

这一提议是一项持续的努力，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 潜在的胰岛素抵抗状态见于II型糖尿病和 肥胖。更直接的目标是确定第三级 转运蛋白的结构与构象动力学 与传输功能相关联。氨基酸序列 这种蛋白质的性质是已知的，这预示着蛋白质是 12个跨膜节段和4个非跨膜节段 亲水性部分。基于这些预测，以下是 提出了实验方案。首先，这些跨膜和 非跨膜片段将被分离和鉴定为 蛋白酶--消化。高效毛细管电泳法分离测定 模型预测的特征性氨基酸含量。这个 这些线段的三维排列将确定 通过用亲脂性共价的交联剂标记它们 探测器和氚水质子。这些方面可能发生的变化 关于移除的三维安排 非跨膜、亲水性片段将由 同样的程序。还将根据以下条件监控这些更改 圆环、二色性、傅里叶变换红外光谱、 辐射灭活靶尺寸与荧光能量传递 测量。底物中的底物结合部位 蛋白质中的识别口袋和转位途径 将通过用光反应底物标记它们来进行研究 类比。侧面特定的、不可运输的模拟和可运输的 类比将被用来区分这些站点。细胞松弛素 B装订袋的特征将通过识别其装订 地点。那些跨膜或非跨膜的亲水性片段 受底物影响或由抑制剂诱导的 构象扰动将通过标记和 鉴定与某些氨基酸反应的氨基酸残基 已知烷基化试剂会使蛋白质功能失活。 并且对底物和抑制剂敏感。四种不同的 烷基化试剂将用于这一目的。最后，作为一个 长期目标，纯化转运蛋白的结晶 将被尝试用于未来的X射线衍射研究。