EFFECT OF APOLIPOPROTEIN STRUCTURAL ADAPTABILITY

本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。研究将集中在这一类蛋白质的关键成员--人类载脂蛋白E(ApoE)。虽然在没有脂的情况下，这种蛋白质的结构信息是可用的，但人们认识到，可交换的载脂蛋白只有在与脂相关的状态下才发挥其生物学作用。有证据表明，这些蛋白质在脂质结合时会发生显著的构象变化)。ApoE的N-末端结构域由一束拉长的两亲性α-螺旋组成。已经提出了一些模型，预测螺旋束可以围绕位于螺旋之间环中的假定铰链域打开。这种构象变化会导致疏水残基的暴露，使它们可以与脂蛋白表面相互作用。有人认为，这种构象变化是可逆的，存在于无脂构象中的螺旋边界保持在与脂结合的状态。脂类结合诱导apoE N-末端结构域构象适应的确切性质将被确定。结构信息将被用于选择用于定点突变的候选氨基酸残基。二硫键工程和荧光共振能量转移将用于评估脂质结合诱导的螺旋重新定位。含有一种色氨酸和一种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距离测量提供了来自激发色氨酸的能量受体。通过在分子中合理地放置能量供体/受体对，将有可能构建脂质结合的螺旋运动图。推测人载脂蛋白E N-末端结构域的构象开放是调节其受体结合活性的重要生理机制。我们将研究脂类相关的全长apoE与培养的人皮肤成纤维细胞上的低密度脂蛋白受体之间的相互作用及其N-末端结构域的构象状态。所获得的结果将为可交换载脂蛋白构象适应性的生理学相关性提供新的信息。

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.