Sahoo D, Narayanaswami V, Kay C M, Ryan R O
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1998 Jan 16;273(3):1403-8. doi: 10.1074/jbc.273.3.1403.
Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an 18-kDa exchangeable apolipoprotein that reversibly associates with lipoprotein particles. In the absence of lipid, apoLp-III exists as an elongated bundle of five amphipathic alpha-helices. Upon lipid association, the protein is postulated to undergo a major conformational change, wherein the bundle opens around hinge loop regions, resulting in exposure of its hydrophobic interior. Fluorescence quenching techniques have been employed to study apoLp-III helix topography and spatial arrangement in phospholipid disc complexes and intact lipoprotein particles. Intrinsic fluorescence of the single tyrosine in apoLp-III was exploited to monitor the location of helix 5 in model disc complexes. To investigate other regions of the protein, site-directed mutagenesis was performed to introduce cysteine residues, replacing Asn-40 (helix 2, N40C) or Leu-90 (helix 3, L90C), thereby providing two mutant apoLp-IIIs, each with a single site for covalent attachment of the extrinsic fluorescent probe, N-(1-pyrene) maleimide. In the lipid-free state, pyrene-N40C- and pyrene-L90C-apoLp-III were highly accessible to the negatively charged aqueous quencher KI, yielding Ksv values of 27.1 and 19.8 M-1, respectively. Upon binding to the surface of a spherical lipoprotein particle, Ksv values for KI decreased by about 90% for both pyrene-labeled apoLp-IIIs, indicating a significant change in the local microenvironment of the fluorophores. A lesser decrease in Ksv was observed when the pyrene-labeled apoLp-IIIs were bound to phospholipid disc complexes. When spin-labeled fatty acids 5-doxylstearic acid and 12-doxylstearic acid were used as lipophilic quenchers, tyrosine and pyrene fluorescence were more effectively quenched by 5-doxylstearic acid in both phospholipid bilayer disc complexes and spherical lipoprotein particles. These data provide insight into the spatial topography of apoLp-III alpha-helices in phospholipid disc complexes and support the concept that interaction with spherical lipoprotein particles results in superficial contact of apoL-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.
来自烟草天蛾（Manduca sexta）的载脂蛋白III（apoLp-III）是一种18 kDa的可交换载脂蛋白，它能与脂蛋白颗粒可逆性结合。在没有脂质的情况下，apoLp-III以由五个两亲性α-螺旋组成的细长束状形式存在。在与脂质结合后，推测该蛋白质会发生重大构象变化，其中束状结构围绕铰链环区域打开，导致其疏水内部暴露。荧光猝灭技术已被用于研究apoLp-III在磷脂盘复合物和完整脂蛋白颗粒中的螺旋拓扑结构和空间排列。利用apoLp-III中单个酪氨酸的固有荧光来监测模型盘复合物中螺旋5的位置。为了研究该蛋白质的其他区域，进行了定点诱变以引入半胱氨酸残基，取代Asn-40（螺旋2，N40C）或Leu-90（螺旋3，L90C），从而得到两种突变型apoLp-III，每种都有一个用于非天然荧光探针N-(1-芘)马来酰亚胺共价连接的单个位点。在无脂质状态下，芘-N40C-和芘-L90C-apoLp-III对带负电荷的水性猝灭剂KI高度可及，其猝灭常数Ksv值分别为27.1和19.8 M-1。当与球形脂蛋白颗粒表面结合时，两种芘标记的apoLp-III的KI的Ksv值均下降约90%，表明荧光团的局部微环境发生了显著变化。当芘标记的apoLp-III与磷脂盘复合物结合时，观察到Ksv的下降较小。当使用自旋标记的脂肪酸5-脱氧硬脂酸和12-脱氧硬脂酸作为亲脂性猝灭剂时，在磷脂双层盘复合物和球形脂蛋白颗粒中，酪氨酸和芘荧光被5-脱氧硬脂酸更有效地猝灭。这些数据为磷脂盘复合物中apoLp-IIIα-螺旋的空间拓扑结构提供了深入了解，并支持了与球形脂蛋白颗粒相互作用导致apoL-III螺旋片段与单层表面进行表面接触的概念，为其可逆结合能力提供了基础。
