School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC 3010, Australia.
Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation, Kirrawee DC, NSW 2232, Australia.
Biochim Biophys Acta Biomembr. 2020 May 1;1862(5):183204. doi: 10.1016/j.bbamem.2020.183204. Epub 2020 Jan 23.
Little is known experimentally about the detailed orientation of membrane-bound maculatin 1.1 (Mac1), an antimicrobial peptide from the skin secretions of Australian tree frogs. In this work multiple N-labelled or H-labelled Mac1 with dodecylphosphocholine (DPC) micelles and isotropic DMPC/DHPC (q = 0.5) bicelles were investigated by solution NMR, circular dichroism (CD) spectroscopy, neutron reflectometry and molecular dynamics (MD) simulations in explicit solvent. In buffer, the N-H HSQC and CD spectra were indicative of the peptide being random coiled. In the presence of micelles or isotropic bicelles, a unique and helical peptide structure that was confirmed by CD was found. The titration of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC solution led to enhanced relaxation of all N labelled residues. The peptide N-terminus was more exposed to Gd-DTPA than the C-terminus in micelles, while only the Gly-4 and Ala-18 resonances were significantly reduced in the presence of isotropic bicelles. MD simulations of Mac1 fully inserted into a DPC micelle converged towards a solvent exposed orientation and a topology where Mac1 was wrapped around the DPC micelle with the more hydrophobic side facing inward. MD simulations of Mac1 fully inserted into a phosphatidylcholine (PC) bilayer converged towards a kinked transmembrane orientation with water molecules penetrating around Lys-8. A deuterium labelled Mac1 used in neutron reflectometry experiments suggested a preferred orientation in zwitterionic PC bilayers. These results give insight into the membrane disrupting activity of Mac1 against cell membranes.
关于澳大利亚树蛙皮肤分泌物中一种抗菌肽 Mac1 的膜结合体的详细取向，实验知之甚少。在这项工作中，使用溶液 NMR、圆二色性（CD）光谱、中子反射计和分子动力学（MD）模拟研究了带有十二烷基磷酸胆碱（DPC）胶束和各向同性 DMPC/DHPC（q = 0.5）双胶束的多个 N 标记或 H 标记的 Mac1。在缓冲液中，N-H HSQC 和 CD 谱表明肽呈无规卷曲。在胶束或各向同性双胶束存在的情况下，发现了一种独特的螺旋肽结构，这一结构通过 CD 得到了证实。将可溶性顺磁试剂钆（Gd-DTPA）滴定到 Mac1-DPC 溶液中，导致所有 N 标记残基的弛豫增强。在胶束中，肽的 N 端比 C 端更暴露于 Gd-DTPA，而仅在各向同性双胶束存在时，Gly-4 和 Ala-18 共振才会显著减少。Mac1 完全插入 DPC 胶束的 MD 模拟收敛到一种暴露在溶剂中的取向和一种拓扑结构，其中 Mac1 缠绕在 DPC 胶束周围，疏水性侧面向内。Mac1 完全插入磷脂酰胆碱（PC）双层的 MD 模拟收敛到一种带有水分子穿透 Lys-8 的扭曲跨膜取向。在中子反射计实验中使用的氘标记 Mac1 表明在两性离子 PC 双层中存在优先取向。这些结果深入了解了 Mac1 对细胞膜的破坏活性。
