EFFECT OF APOLIPOPROTEIN STRUCTURAL ADAPTABILITY

载脂蛋白结构适应性的影响

• 批准号: 6040851
• 负责人: ROBERT O'Mara RYAN
• 金额: $ 34.54万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6040851
• 关键词: X ray crystallography; apolipoprotein E; blood lipoprotein metabolism; chromophore; conformation; cysteine; disulfide bond; fibroblasts; fluorescence resonance energy transfer; homeostasis; human tissue; intermolecular interaction; low density lipoprotein receptor; mutant; protein binding; protein isoforms; protein structure function; receptor binding; site directed mutagenesis; tissue /cell culture; tryptophan

The long terms goal of this research is to elucidate the metabolic significance of exchangeable apolipoprotein structural alterations. Studies will focus on a key member of this protein class, human apolipoprotein E (apoE). While structural information is available for this protein in the absence of lipid, it is recognized that exchangeable apolipoproteins exert their biological effects only in a lipid-associated state. Evidence suggests these proteins undergo significant conformational changes) upon lipid binding. The N-terminal domain of apoE is organized as a bundle of elongated amphipathic alpha-helices. Models have been proposed which predict the helix bundle can open about a putative hinge domain located in the loop between helices. Such a conformational change would result in exposure of hydrophobic residues, making them available for interaction with lipoprotein surfaces. It is proposed that this conformational change is reversible and that helix boundaries present in the lipid-free conformation are maintained in the lipid associated state. The precise nature of lipid binding-induced conformational adaptations of apoE N-terminal domain will be determined. Structural information will be used to select candidate amino acid residues for site directed mutagenesis. Disulfide bond engineering and fluorescence resonance energy transfer will be performed to evaluate lipid binding-induced helix repositioning. Mutant proteins containing a single tryptophan and a single cysteine will be expressed in bacteria. Modification of cysteine with an appropriate chromophore provides an energy acceptor from excited tryptophan for distance measurements. Through judicious placement of energy donor/acceptor pairs in the molecule, it will be possible to construct a map of helix movements upon lipid binding. It is hypothesized that conformational opening of the N-terminal domain of human apoE represents a physiologically important mechanism for regulation of its receptor binding activity. Studies will be performed to characterize the correlation between lipid-associated full length apoE interactions with the low density lipoprotein receptor on cultured human skin fibroblasts and the conformational status of its N-terminal domain. The results obtained will provide new information about the physiological relevance of the conformational adaptability of exchangeable apolipoproteins.

这项研究的长期目标是阐明可交换载脂蛋白结构改变的代谢意义。 研究将重点放在该蛋白质类的关键成员，即人载脂蛋白E（APOE）。 尽管在没有脂质的情况下可用于该蛋白质的结构信息，但人们认识到，可兑换的载脂蛋白仅在脂质相关状态下发挥其生物学作用。证据表明这些蛋白质在脂质结合后经历了显着的构象变化。 APOE的N末端结构域被组织为一束伸长的两亲性α-螺旋。 已经提出了模型，该模型可以预测螺旋束可以打开有关位于螺旋之间环路中的假定铰链结构域。 这种构象变化将导致疏水残基暴露，使其与脂蛋白表面相互作用。 有人提出，这种构象变化是可逆的，并且在脂质相关状态中保持无脂质构象中存在的螺旋边界。将确定脂质结合诱导的APOE N末端结构域的构象适应的确切性质。结构信息将用于选择候选氨基酸残基，以定向诱变。 将进行二硫键工程和荧光共振能量转移，以评估脂质结合诱导的螺旋重新定位。含有单色氨酸和单一半胱氨酸的突变蛋白将在细菌中表达。 用适当的发色团修饰半胱氨酸为激发色氨酸提供了能量受体，以进行距离测量。 通过在分子中明智地放置能量供体/受体对，可以在脂质结合上构造螺旋运动图。 假设人apoE的N末端结构域的构象打开代表了调节其受体结合活性的生理重要机制。 将进行研究以表征脂质相关的全长APOE与低密度脂蛋白受体在培养的人皮肤成纤维细胞上与其N末端结构域的构象状态之间的相关性。 获得的结果将提供有关可交换载脂蛋白构象适应性的生理相关性的新信息。