Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.
Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.
复制标题
通过氨基酸取代和三甲胺-N-氧化物探测人载脂蛋白 C-1 的构象。
DOI：
10.1110/ps.8.10.2055
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Gursky,O
中科院分区：
文献类型：
--
作者：
Gursky,O
为了在单个氨基酸水平上测试可交换载脂蛋白在水溶液中和在渗透剂三甲胺-N-氧化物（TMAO）存在下的构象，用圆二色性（CD）分析了6个在预测的α-螺旋区含有点突变的人载脂蛋白C-1（apoC-1，57个残基）的合成肽类似物。在中性低盐溶液中，单体野生型和血浆apoC-1的CD光谱和熔解曲线显示，在22 °C时，31 ± 4%的α-螺旋结构可逆熔解，Tm，WT = 50 ± 2 °C，范特霍夫焓ΔHv，WT（Tm）= 18 ± 2 kcal/mol。G15 A取代导致α-螺旋含量增加42 ± 4%，Tm增加，G15 A = 57 ± 2 °C，这对应于δΔGapp = +0.4 ± 1.5 kcal/mol的稳定性。G15 P突变体在22 °C下具有约20%的α-螺旋含量，并且在加热至90 °C时以低协同性展开。R23 P和T45 P突变体在0-90 °C下完全解折叠。相反，Q31 P突变导致不稳定或解折叠。因此，R23和T45位置对于apoC-1单体中的合作α-螺旋单元的稳定性是必不可少的，G15位于其外围，Q31位于非螺旋连接区。我们的研究结果表明，亲诱变加上CD提供了一种工具，用于分配的二级结构的蛋白质组，这应该是有用的其他自缔合蛋白质，不适合在水溶液中的NMR结构分析。TMAO诱导apoC-1发生可逆的螺旋-卷曲转变，α-螺旋含量最高可达74%。与在脂质结合的apoC-1中观察到的最大α-螺旋含量73%相比，TMAO稳定的二级结构类似于功能性脂质结合的载脂蛋白构象。
To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted α-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 ± 4% α-helical structure at 22 °C that melts reversibly with Tm,WT = 50 ± 2 °C and van't Hoff enthalpy ΔHv,WT(Tm) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α-helical content of 42 ± 4% and an increased Tm,G15A = 57 ± 2 °C, which corresponds to stabilization by δΔGapp = +0.4 ± 1.5 kcal/mol. G15P mutant has ∼20% α-helical content at 22 °C and unfolds with low cooperativity upon heating to 90 °C. R23P and T45P mutants are fully unfolded at 0–90 °C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative α-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal α-helical content reaching 74%. Comparison with the maximal α-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.