McLean L R, Hagaman K A, Owen T J, Payne M H, Davidson W S, Krstenansky J L
Marion Merrell Dow Research Institute, Cincinnati, OH 45215.
Biochim Biophys Acta. 1991 Oct 15;1086(1):106-14. doi: 10.1016/0005-2760(91)90161-a.
To examine the relationship between peptide sequence and the interaction of amphipathic alpha-helical peptides with phosphatidylcholines, various methods of mixing the peptide and lipid were explored. A series of amphipathic alpha-helical peptides containing from 10 to 18 residues were synthesized by solid-phase techniques. An 18-residue peptide and two relatively hydrophobic 10-residue peptides did not disrupt dimyristoylphosphatidylcholine liposomes when added to the lipid in buffer. However, when the peptides were premixed with lipid in a suitable organic solvent and then reconstituted with aqueous buffer, clear micelles were formed, indicating association of the amphipathic alpha-helical peptide with lipid. In general, the best solvent for this purpose was trifluoroethanol. The circular dichroic and fluorescence spectra of peptides which readily formed clear mixtures when mixed in buffer with dimyristoylphosphatidylcholine liposomes were similar when prepared either by the alternative pathway technique using trifluoroethanol or by a cholate removal technique. For the peptides which did not clear liposomes in buffer, first mixing with dimyristoylphosphatidylcholine in trifluoroethanol resulted in an increase in the alpha-helicity of the peptides as judged by circular dichroic spectra and a blue-shift in the fluorescence emission maxima of the single tryptophan residue in each peptide. These data are consistent with formation of an amphipathic alpha-helix in lipid by peptides which based on mixing experiments with dimyristoylphosphatidylcholine liposomes in buffer at the phase transition temperature of the lipid would be considered ineffective in lipid binding. Thus, simple mixing of peptides with liposomes may give misleading results concerning the intrinsic affinity of a particular peptide sequence for lipid. In addition, the data demonstrate that relatively hydrophobic amphipathic alpha-helical peptides which do not form small micelles with dimyristoylphosphatidylcholine spontaneously in aqueous solution may interact with lipid as typical amphipathic alpha-helices when mixed by an alternative pathway.
为了研究肽序列与两亲性α-螺旋肽与磷脂酰胆碱相互作用之间的关系，探索了多种混合肽和脂质的方法。通过固相技术合成了一系列含10至18个残基的两亲性α-螺旋肽。当将一个18个残基的肽和两个相对疏水的10个残基的肽添加到缓冲液中的脂质中时，它们不会破坏二肉豆蔻酰磷脂酰胆碱脂质体。然而，当将这些肽在合适的有机溶剂中与脂质预混合，然后用水性缓冲液重构时，会形成清晰的胶束，这表明两亲性α-螺旋肽与脂质发生了缔合。一般来说，用于此目的的最佳溶剂是三氟乙醇。当通过使用三氟乙醇的替代途径技术或通过胆酸盐去除技术制备时，在缓冲液中与二肉豆蔻酰磷脂酰胆碱脂质体混合时容易形成清晰混合物的肽的圆二色光谱和荧光光谱相似。对于在缓冲液中不能使脂质体澄清的肽，首先在三氟乙醇中与二肉豆蔻酰磷脂酰胆碱混合，通过圆二色光谱判断，肽的α-螺旋度增加，并且每个肽中单个色氨酸残基的荧光发射最大值发生蓝移。这些数据与肽在脂质中形成两亲性α-螺旋一致，基于在脂质的相变温度下在缓冲液中与二肉豆蔻酰磷脂酰胆碱脂质体的混合实验，这些肽在脂质结合方面被认为是无效的。因此，肽与脂质体的简单混合可能会在特定肽序列对脂质的内在亲和力方面产生误导性结果。此外，数据表明，在水溶液中不能与二肉豆蔻酰磷脂酰胆碱自发形成小胶束的相对疏水的两亲性α-螺旋肽，当通过替代途径混合时，可能会作为典型的两亲性α-螺旋与脂质相互作用。
