Sparks D L, Davidson W S, Lund-Katz S, Phillips M C
Department of Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
J Biol Chem. 1993 Nov 5;268(31):23250-7.
The effects of cholesterol on the conformation and net charge of apoA-I have been investigated in homogeneous recombinant high density lipoprotein (HDL) particles. ApoA-I charge and structure in discoidal recombinant HDL complexes containing palmitoyloleoylphosphatidylcholine and cholesterol have been quantitated by guanidine HCl denaturation, circular dichroism, electrokinetic analysis, and NMR spectroscopy of [13C]lysine-labeled apoA-I. In a discoidal particle containing 2 molecules of apoA-I and 160 molecules of palmitoyloleoylphosphatidylcholine, apoA-I exhibits an alpha-helix content of 75%, and the particle has a net negative surface charge of -5.2e/mol of apoA-I at pH 8.6. Addition of 2 molecules of cholesterol to this complex has no significant effect upon particle size, but slightly decreases the net charge (-5.0e) and alpha-helix content (68%) of apoA-I and enhances the stability of the helical segments, as reflected by an increase in the free energy of unfolding from 2.9 to 3.5 kcal/mol. In contrast, increasing the cholesterol content to 20 molecules/particle progressively increases particle size and apoA-I net negative charge (-6.1e), and there is a concomitant reduction in the free energy of stabilization of the alpha-helical structure in apoA-I to 2.2 kcal/mol. (13CH3)2-Lys resonances from apoA-I in discoidal recombinant HDL exhibit six chemical shifts at pH 10; these peaks originate from dimethyl-Lys residues that have pKa values ranging from 8.4 to 10.3. The titration behavior of apoA-I Lys residues is generally similar in the presence and absence of cholesterol, except that 4 Lys residues titrate at a significantly higher pH in the presence of cholesterol. These data are consistent with cholesterol having a direct effect on apoA-I conformation and charge in HDL. Structural changes of this magnitude can affect the interactions between HDL and various plasma proteins and cell surfaces. It is therefore likely that the cholesterol content of HDL plays an important role in regulating the metabolism of this lipoprotein.
在均一的重组高密度脂蛋白（HDL）颗粒中，研究了胆固醇对载脂蛋白A-I（apoA-I）构象和净电荷的影响。通过盐酸胍变性、圆二色性、电动分析以及对[13C]赖氨酸标记的apoA-I进行核磁共振光谱分析，对含有棕榈油酰油酰磷脂酰胆碱和胆固醇的盘状重组HDL复合物中的apoA-I电荷和结构进行了定量。在一个含有2个apoA-I分子和160个棕榈油酰油酰磷脂酰胆碱分子的盘状颗粒中，apoA-I的α-螺旋含量为75%，在pH 8.6时，该颗粒的净表面电荷为-5.2e/mol的apoA-I。向该复合物中添加2个胆固醇分子对颗粒大小没有显著影响，但略微降低了apoA-I的净电荷（-5.0e）和α-螺旋含量（68%），并增强了螺旋段的稳定性，这表现为解折叠自由能从2.9千卡/摩尔增加到3.5千卡/摩尔。相比之下，将胆固醇含量增加到20个分子/颗粒会逐渐增加颗粒大小和apoA-I的净负电荷（-6.1e），同时apoA-I中α-螺旋结构的稳定自由能降低到2.2千卡/摩尔。盘状重组HDL中apoA-I的(13CH3)2-Lys共振在pH 10时呈现六个化学位移；这些峰来自pKa值范围为8.4至10.3的二甲基-Lys残基。apoA-I赖氨酸残基的滴定行为在有胆固醇和无胆固醇的情况下通常相似，只是有4个赖氨酸残基在有胆固醇存在时在显著更高的pH下滴定。这些数据与胆固醇对HDL中apoA-I构象和电荷有直接影响一致。这种程度的结构变化会影响HDL与各种血浆蛋白和细胞表面之间的相互作用。因此，HDL中的胆固醇含量可能在调节这种脂蛋白的代谢中起重要作用。
