Leroy A, Jonas A
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign 61801.
Biochim Biophys Acta. 1994 Jun 2;1212(3):285-94. doi: 10.1016/0005-2760(94)90202-x.
The effect of n-propanol on the secondary and tertiary structure of human apolipoprotein A-I (apoA-I), an interfacial protein, was investigated using near and far ultraviolet (UV)-circular dichroism (CD) and fluorescence spectroscopy, as well as limited proteolytic digestion with trypsin, and cross-linking with bis(sulfosuccinimidyl) suberate. The structure of apoA-I in n-propanol (30%, v/v) was compared with that in Tris buffer and in reconstituted, spherical or discoidal, high density lipoproteins (rHDL). Addition of n-propanol to apoA-I in Tris buffer induces major changes in its near and far CD spectra: alpha-helical content increases by 27% and the near UV-CD spectrum becomes very similar to that of apoA-I in rHDL particles. Fluorescence spectral, lifetime, and polarization results, and quenching by KI confirm that major structural changes occur in the N-terminal half of apoA-I as n-propanol is added: the Trp residues become more exposed to solvent than in buffer alone or in rHDL. Higher concentrations of guanidine hydrochloride or urea are required to denature apoA-I in n-propanol than in buffer alone, but a similar free energy of unfolding is observed. The N-terminus of apoA-I is relatively resistant to trypsin digestion and the C-terminus has equivalent digestion sites for apoA-I in the three states, but the kinetics of digestion are much slower in n-propanol and in rHDL compared to apoA-I in Tris buffer. Cross-linking experiments reveal that dimers of apoA-I exist in n-propanol, in contrast to dimers plus multimeric aggregates in Tris buffer. From these results we conclude that in 30% n-propanol the structure of apoA-I approaches that of 'native' lipid-bound apoA-I, in contrast to its structure in the aqueous Tris buffer.
使用近紫外和远紫外圆二色光谱（CD）、荧光光谱、胰蛋白酶有限水解以及与辛二酸双（磺基琥珀酰亚胺酯）交联的方法，研究了正丙醇对人载脂蛋白A-I（apoA-I，一种界面蛋白）二级和三级结构的影响。将apoA-I在正丙醇（30%，v/v）中的结构与在Tris缓冲液以及重组的球形或盘状高密度脂蛋白（rHDL）中的结构进行了比较。向Tris缓冲液中的apoA-I添加正丙醇会使其近紫外和远紫外CD光谱发生重大变化：α-螺旋含量增加27%，近紫外CD光谱变得与rHDL颗粒中的apoA-I非常相似。荧光光谱、寿命和偏振结果以及KI淬灭证实，随着正丙醇的添加，apoA-I的N端发生了重大结构变化：与单独在缓冲液中或在rHDL中相比，色氨酸残基对溶剂的暴露程度更高。与单独在缓冲液中相比，使正丙醇中的apoA-I变性需要更高浓度的盐酸胍或尿素，但观察到相似的解折叠自由能。apoA-I的N端对胰蛋白酶消化相对抗性较强，C端在三种状态下对apoA-I具有等效的消化位点，但与Tris缓冲液中的apoA-I相比，正丙醇和rHDL中的消化动力学要慢得多。交联实验表明，与Tris缓冲液中存在二聚体和多聚体聚集体不同，apoA-I在正丙醇中以二聚体形式存在。从这些结果我们得出结论，与在水性Tris缓冲液中的结构相比，在30%正丙醇中apoA-I的结构接近“天然”脂质结合的apoA-I的结构。
