Determining structural changes in model membranes of Gram-negative bacteria during their interaction with antimicrobial peptides: spectroscopic and microscopic study under electrochemical control

抗菌肽（Antimicrobial Peptides，AMP）是一类带正电荷的小分子多肽，它与微生物细胞膜相互作用，导致细胞膜结构的改变和细胞死亡。相比之下，AMP与宿主生物体细胞的相互作用既不影响膜的完整性也不影响结构。在分子水平上识别参与这种相互作用的脂质和肽的变化对于理解肽对微生物的选择性、先天免疫系统中发生的反应以及开发新一代抗生素至关重要。制造的模型的脂质膜的病原体和宿主生物体耦合到原位结构分析技术可能有助于我们了解这种相互作用的分子方面和特异性的抗菌肽的某些膜。由于细菌细胞膜的组成和超分子结构与哺乳动物细胞膜有很大的不同。利用Langmuir-Blodgett转移法可以制备出与革兰氏阴性菌的组成和结构非常接近的高度不对称的脂质双层。富含磷脂的对称和不对称双层将被用作哺乳动物细胞膜的模型。本研究将使用天蚕素和蜂毒肽。静电相互作用在维持细菌细胞膜的结构以及AMP与细胞表面的结合中起重要作用。在这种相互作用的结果中，膜的电化学性质改变。脂质双层在电极表面上的沉积允许原位监测膜的电化学性质的变化，由于其与AMP的相互作用。为了确定膜结构的原位变化，将使用偏振调制红外反射吸收光谱和电化学控制下的扫描力显微镜。所提出的方法将提供，在亚分子水平上，在模型膜的每个脂质层的极性和疏水部分的取向，构象和水合作用的信息，以及作为作用在膜上的电场的函数的相互作用肽的结构和取向。通过这种方式，将提供暴露于生理电场的脂质膜结构变化的整体图片，旨在阐明抗菌肽与细胞膜相互作用的分子方面。

Antimicrobial peptides (AMPs) are small positively charged polypeptides which interact with microbial cell membranes leading to a distortion of the membrane structure and cell death. By contrast, the interaction of AMPs with cell of a host organism affects neither the membrane integrity nor structure. The recognition at the molecular level of changes at lipids and peptides involved in this interaction is crucial to understand the selectivity of peptides to microbes, reactions occurring in the innate immune system and to develop a new generation of antibiotics. Fabrication of models of lipid membranes of pathogens and of host organisms coupled to in situ structure analyzing techniques may help us to understand molecular aspects of this interaction and the specificity of antimicrobial peptides to certain membranes. Since the composition and supramolecular structure of bacterial cell membranes differs significantly from that of mammalian membranes. Highly asymmetric lipid bilayers mimicking closely the composition and structure of Gram-negative bacteria will be produced using Langmuir-Blodgett transfer. Phospholipid rich symmetric and asymmetric bilayers will be used as models of mammalian cell membranes. Cecropin and melittin peptides will be used in this study. Electrostatic interactions play an important role in the maintenance of the structure of bacterial cell membranes as well as in binding of an AMP to the cell surface. In the result of this interaction the electrochemical properties of the membrane change. A deposition of the lipid bilayer on the electrode surface allows in situ monitoring of changes of the electrochemical properties of the membrane due to its interaction with AMPs. In order to determine in situ changes in the membrane structure polarization modulation infrared reflection absorption spectroscopy and scanning force microscopy under electrochemical control will be used. The proposed approach will provide, at sub-molecular level, information on the orientation, conformation and hydration of polar and hydrophobic parts of each lipid layer of model membranes as well as the structure and orientation of the interacting peptide as a function of electric field acting at the membrane. In this way a holistic picture of structural changes in lipid membranes exposed to physiological electric fields will be provided aiming at the elucidation of molecular aspects of antimicrobial peptides interactions with cell membranes.