Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.
Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.
复制标题
重组高密度脂蛋白盘中载脂蛋白 A-I 的排列：基于荧光共振能量转移实验的替代模型。
作者：
Tricerri,MA;BehlingAgree,AK;Sanchez,SA;Bronski,J;Jonas,A
载脂蛋白A-I （apoA-I）在盘状高密度脂蛋白（HDL）与磷脂复合物中的折叠和组织尚未完全解决。近20年来，人们普遍认为apoA-I的两亲螺旋平行于磷脂的酰基链（“尖桩栅栏”模型）。然而，基于一个大的无脂片段的apoA-I的x射线晶体结构，最近提出了一个“带模型”。在这个模型中，两个反平行的apoa - 1分子的螺旋呈圆形排列，并垂直于磷脂酰基链。为了获得apoA-I在盘状HDL中的空间组织的结结性信息，我们设计了三种不同的apoA-I半胱氨酸突变体（D9C, A124C, A232C），用荧光探针ALEXA-488或ALEXA-546（荧光素和罗丹明衍生物）进行特异性标记。标记的apoa - 1被重组成包含两个蛋白质分子和双棕榈酰磷脂酰胆碱的明确的HDL复合物，并在三个定量荧光共振能量转移（FRET）实验中使用这些复合物来确定HDL颗粒中两个特定位点之间的距离。将FRET测量的距离（4.7 ~ 7.8 nm）与现有模型预测的距离进行比较，表明桩栅模型和带模型都不能解释实验结果；相反，距离和寿命数据强烈地暗示了每个apoA-I单体的发夹折叠，大多数螺旋垂直于磷脂酰基链，以及HDL颗粒中两个apoA-I分子的随机首尾和首尾排列。
The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids (“picket fence” model). However, based on the X-ray crystal structure of a large, lipid-free fragment of apoA-I, a “belt model” was recently proposed. In this model, the helices of two antiparallel apoA-I molecules are extended in a circular arrangement and lie perpendicular to the phospholipid acyl chains. To obtain conclusive information on the spatial organization of apoA-I in discoidal HDL, we engineered three separate cysteine mutants of apoA-I (D9C, A124C, A232C) for specific labeling with the fluorescence probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). The labeled apoA-I was reconstituted into well-defined HDL complexes containing two molecules of protein and dipalmitoylphosphatidylcholine, and the complexes were used in three quantitative fluorescence resonance energy transfer (FRET) experiments to determine the distances between two specific sites in an HDL particle. Comparison of the distances measured by FRET (4.7−7.8 nm) with those predicted from the existing models indicated that neither the picket fence nor the belt model can account for the experimental results; rather, a hairpin folding of each apoA-I monomer with most helices perpendicular to the phospholipid acyl chains and a random head-to-tail and head-to-head arrangement of the two apoA-I molecules in the HDL particles are strongly suggested by the distance and lifetime data.