LIPID BINDING PROTEINS WITH ALTERED SPECIFICITY

特异性改变的脂质结合蛋白

• 批准号: 2148081
• 负责人: DAVID PAUL CISTOLA
• 金额: $ 20.06万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2148081
• 关键词: all trans retinol; biophysics; calorimetry; chemical binding; chimeric proteins; cholanate compound; cholesterol; circular dichroism; computer simulation; conformation; fatty acid binding protein; fatty acids; fluorescence spectrometry; intermolecular interaction; ligands; mutant; nuclear magnetic resonance spectroscopy; protein engineering; protein purification; protein structure function; retinoid binding proteins; site directed mutagenesis; stoichiometry; thermodynamics

Absorptive enterocytes in the small intestine contain four homologous lipid-binding proteins which represent over 5% of the total soluble cellular protein. These proteins are termed ileal lipid-binding protein, intestinal fatty acid-binding protein, "liver" fatty acid-binding protein, and cellular retinol-binding protein II. They are proposed to serve distinct functional roles in absorption, transport and metabolism of different classes of dietary and biliary lipids. Because of their different specificities, these proteins may ultimately represent excellent targets for the rational design of agents to selectively reduce the intestinal absorption of saturated and unsaturated fatty acids, bile salts, and cholesterol. However, the factors that control molecular recognition in this family of proteins has not yet been established. The main objective of this research is to identify the structural, dynamic and energetic determinants of ligand-binding affinity and specificity for the four distinct lipid-binding proteins from the small intestine. The specific aims of the proposed research are: (i) to characterize the binding pro mutant and chimeric lipid- binding proteins, (ii) to establish sequence-specific 1H, 13C, and 15N NMR assignments and determine the solution structures for several mutant proteins with altered ligand specificities, and (iii) to design and synthesize novel ligands that bind with high affinity to act as inhibitors. The feasibility of the proposed experiments is firmly established by preliminary results, including the high-yield expression and purification of natural abundance and isotope-enriched proteins, the development of biophysical assays for probing lipid-protein interactions, the generation of mutants with altered specificity, and the collection and assignment of triple-resonance 3-D NMR data with excellent sensitivity and resolution. A high intake of dietary fat has been associated with an increased risk of atherosclerosis and colon cancer. A detailed knowledge of the molecular factors governing intestinal fat absorption and homeostasis will ultimately lead to more precise dietary recommendations and novel pharmacologic strategies to prevent these prevalent and life-threatening diseases. In particular, a detailed study of the molecular determinants of lipid-binding specificity, stoichiometry and affinity in the lipid-binding proteins from intestine will contribute fundamentally important information for this purpose.

小肠中的吸收性肠细胞包含四个 同源脂质结合蛋白，代表超过5％ 总可溶性细胞蛋白。这些蛋白质称为回肠 脂质结合蛋白，肠道脂肪酸结合蛋白， “肝”脂肪酸结合蛋白和细胞视黄醇结合 蛋白II。提议他们在 不同类别的吸收，运输和代谢 饮食和胆道脂质。因为他们的不同 这些蛋白质特异性最终可能代表了极好的 代理人合理设计的目标，有选择地减少 饱和和不饱和脂肪酸的肠吸收， 胆汁和胆固醇。但是，控制的因素 该蛋白质家族中的分子识别尚未 已确立的。 这项研究的主要目的是确定结构性， 配体结合亲和力的动态和能量决定因素和 来自四种不同的脂质结合蛋白的特异性 小肠。拟议研究的具体目的是： （i）表征结合pro突变体和嵌合脂质 结合蛋白，（ii）建立序列特异性1H，13C和 15N NMR分配并确定解决方案结构 几种具有改变配体特异性的突变蛋白，并且 （iii）设计和合成与高结合的新型配体 亲和力充当抑制剂。提议的可行性 实验是通过初步结果牢固确定的，包括 自然丰度的高收益表达和纯化 富含同位素的蛋白质，生物物理测定的发展 为了探测脂质 - 蛋白质相互作用，突变体的产生 随着特异性的改变以及收集和分配 具有出色灵敏度和 解决。 饮食脂肪的高摄入量与增加有关 动脉粥样硬化和结肠癌的风险。详细了解 控制肠道脂肪的分子因素和 稳态最终将导致更精确的饮食 建议和新颖的药理学策略，以防止这些策略 流行和威胁生命的疾病。特别是详细的 研究脂质结合特异性的分子决定因素， 脂质结合蛋白的化学计量和亲和力 肠将为根本重要的信息贡献 这个目的。