Orädd Greger, Schmidtchen Artur, Malmsten Martin
Department of Biophysical Chemistry, Umeå University, SE-90187, Umeå, Sweden.
Biochim Biophys Acta. 2011 Jan;1808(1):244-52. doi: 10.1016/j.bbamem.2010.08.015. Epub 2010 Aug 27.
Effects of peptide hydrophobicity on lipid membrane binding, incorporation, and defect formation was investigated for variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQHARASHLGLAR), in anionic 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes. Using a method combination of, e.g., ellipsometry, CD, and fluorescence spectroscopy, it was shown that peptide adsorption, as well as peptide-induced liposome leakage and bactericidal potency against Escherichia coli and Pseudomonas aeruginosa, was promoted by increasing the hydrophobicity of CNY21 through either substituting the two histidines (H) in CNY21 with more hydrophobic leucine (L) residues, or end-tagging with tritryptophan (WWW). Fluorescence spectroscopy revealed that both CNY21WWW and the WWW tripeptide localized to the polar headgroup region of these phospholipid membranes. Deuterium NMR experiments on macroscopically oriented membranes containing fully (palmitoyl) deuterated POPC (POPC-d(31)) demonstrated that both CNY21L and CNY21WWW induced disordering of the lipid membrane. In contrast, for cholesterol-supplemented POPC-d(31) bilayers, peptide-induced disordering was less pronounced in the case of CNY21L, indicating that the peptide is unable to partition to the interior of the lipid membrane in the presence of cholesterol. CNY21WWW, on the other hand, retained its membrane-disordering effect also for cholesterol-supplemented POPC-d(31). These findings were supported by pulsed field gradient NMR experiments where the lateral lipid diffusion was determined in the absence and presence of peptides. Overall, the results provide some mechanistic understanding to previously observed effects of peptide hydrophobization through point mutations and end-tagging, particularly so for complement-based antimicrobial peptides.
研究了补体衍生抗菌肽CNY21（CNYITELRRQHARASHLGLAR）变体的肽疏水性对脂质膜结合、掺入和缺陷形成的影响，实验采用阴离子型1-棕榈酰-2-油酰磷脂酰乙醇胺（POPE）/1-棕榈酰-2-油酰磷脂酰甘油（POPG）和两性离子型1-棕榈酰-2-油酰磷脂酰胆碱（POPC）膜。通过椭圆偏振法、圆二色光谱法和荧光光谱法等方法组合表明，通过将CNY21中的两个组氨酸（H）替换为疏水性更强的亮氨酸（L）残基，或用三色氨酸（WWW）进行末端标记来增加CNY21的疏水性，可促进肽吸附以及肽诱导的脂质体泄漏和对大肠杆菌和铜绿假单胞菌的杀菌效力。荧光光谱显示，CNY21WWW和三肽WWW均定位于这些磷脂膜的极性头部区域。对含有完全（棕榈酰）氘代POPC（POPC-d(31)）的宏观取向膜进行的氘核磁共振实验表明，CNY21L和CNY21WWW均诱导脂质膜无序化。相比之下，对于添加胆固醇的POPC-d(31)双层膜，在CNY21L的情况下，肽诱导的无序化不太明显，这表明在存在胆固醇的情况下，该肽无法分配到脂质膜内部。另一方面，CNY21WWW对添加胆固醇的POPC-d(31)也保留了其膜无序化作用。这些发现得到了脉冲场梯度核磁共振实验的支持，该实验在不存在和存在肽的情况下测定了横向脂质扩散。总体而言，这些结果为先前观察到的通过点突变和末端标记进行肽疏水化的影响提供了一些机制理解，特别是对于基于补体的抗菌肽。
