Tang Yan-Chun, Deber Charles M
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada.
Biopolymers. 2004;76(2):110-8. doi: 10.1002/bip.10566.
Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK-NH(2) has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an alpha-helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane-permeable character by replacing most Lys residues with uncharged peptoid [N-(R)glycyl] residues, which might similarly confer water solubility while retaining membrane-interactive properties of the hydrophobic core. Several "peptoid-tagged" derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water-soluble peptides retain the alpha-helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix-breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid-tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water-solubilizing tags and, as such, provide a potentially valuable feature of design of membrane-interactive peptides.
已证明对母序列KKAAALAAAAALAAWAALAAAKKKK-NH(2)的疏水肽进行赖氨酸标记可通过增溶作用促进其合成和纯化，但对于其以α-螺旋构象插入膜中而言，不会影响疏水核心序列的内在特性。然而，由于这些肽带正电荷，它们常常会与膜表面的磷脂头部基团结合，这会抑制它们跨双层插入和/或阻止它们跨细胞双层运输。我们试图通过用不带电荷的类肽[N-(R)甘氨酰]残基取代大多数赖氨酸残基来开发具有更多中性且膜通透性的肽，这可能同样赋予水溶性，同时保留疏水核心的膜相互作用特性。制备了具有不同类肽含量的母肽的几种“类肽标记”衍生物，六个赖氨酸残基中的五个被类肽Nala和/或Nval取代。通过圆二色光谱法测量这些肽的构象表明，尽管已知类肽标签具有破坏螺旋的能力，但这些水溶性肽在胶束（溶血磷脂酰胆碱和十二烷基硫酸钠）中仍保留α-螺旋结构。色氨酸荧光光谱的蓝移和用丙烯酰胺进行的猝灭实验证实，类肽标记的肽可自发插入胶束膜中。结果表明，引入不带电荷的标签后，膜与肽之间的相互作用主要由肽核心的疏水性决定，而非赖氨酸与脂质头部基团之间的静电相互作用。总体研究结果表明，类肽残基作为不带电荷的增溶标签是赖氨酸有效的替代物，因此为膜相互作用肽的设计提供了一个潜在的有价值的特性。
