Subbarao N K, Fielding C J, Hamilton R L, Szoka F C
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143.
Proteins. 1988;3(3):187-98. doi: 10.1002/prot.340030307.
The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.
塞格雷斯特及其同事提出的两亲性螺旋理论（《欧洲生物化学学会联合会快报》：38：247 - 253，1974年）认为，血清载脂蛋白的脂质结合片段呈α螺旋构象。此外，这些螺旋具有一个疏水面和一个亲水面，且带有正电荷和负电荷残基的特定分布。这些带电荷残基的模式在脂质结合以及片段对卵磷脂胆固醇酰基转移酶（LCAT）的激活作用中的重要性仍存在争议。我们设计了一种由30个残基组成的肽，即GALA，它在α螺旋构象下具有一个由谷氨酸残基组成的亲水表面（萨巴拉奥等人：《生物化学》26：2964 - 2972，1987年）。在中性pH条件下，GALA与二肉豆蔻酰磷脂酰胆碱（DMPC）相互作用时，其行为类似于血清载脂蛋白；GALA的氨基末端色氨酸在其荧光发射光谱中发生蓝移，并且圆二色性（CD）光谱表明GALA在DMPC存在时获得了α螺旋结构。通过凝胶渗透色谱法可以分离出一种DMPC - GALA：19/1（摩尔比）的复合物。这种复合物具有盘状结构，其边缘厚度约为44埃，直径为170至350埃。GALA以DMPC为底物时可激活LCAT，但以不饱和磷脂为底物时则不能。GALA在DMPC囊泡中的表观分配系数比在卵磷脂囊泡中的大100倍。在中性pH条件下，GALA与不饱和脂质的相互作用非常微弱，以至于在此处使用的条件下，无法检测到GALA光谱性质或脂质体结构的可检测变化。GALA的序列与先前研究的Apo A1模型肽不同，其亲水面上没有带正电荷的残基。这表明，在与饱和磷脂形成盘状结构或用此类脂质底物激活LCAT时，Apo A1样肽中的正电荷并非必需。
