Wieprecht T, Dathe M, Epand R M, Beyermann M, Krause E, Maloy W L, MacDonald D L, Bienert M
Forschungsinstitut für Molekulare Pharmakologie, Alfred Kowalke Strasse 4, D-10315 Berlin, Germany.
Biochemistry. 1997 Oct 21;36(42):12869-80. doi: 10.1021/bi971398n.
To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.
为了研究带正电荷的螺旋面所张角度对膜活性的影响，从抗菌肽马盖宁2的序列出发，设计了六种两亲性α - 螺旋肽，其角度在80度至180度之间，但保留了疏水性、疏水矩和正电荷总量。圆二色性研究表明，所有类似物在囊泡悬浮液中均呈α - 螺旋构象。肽从带负电荷的磷脂酰甘油（PG）囊泡中诱导染料释放的能力随角度增加而降低。然而，带正电荷残基角度大（140 - 180度）的肽在两性离子磷脂酰胆碱（PC）和混合PC/PG（3:1）囊泡上表现出比角度小（80 - 120度）的类似物更高的通透活性。此外，大角度类似物在抗菌和溶血试验中更具活性。这些类似物的抗菌特异性降低。结合研究表明，大角度和高含量的带负电荷磷脂有利于肽的结合。相反，小角度和低负膜电荷增强了结合肽部分的膜通透效率。所有类似物都使磷脂酰乙醇胺的双层相相对于反相六角相更稳定。因此，可以排除L类通透机制。此外，类似物不是通过诱导正曲率应变或“地毯样”机制起作用。我们的结果符合孔道机制：带正电荷残基角度增大的类似物的膜通透效率因孔道内相邻螺旋之间的静电排斥而降低，从而导致成孔概率降低和/或孔道不稳定。
