Probing the dynamics, orientation and conformation of lipopolysaccharide in bacterial membranes through bioorthogonal chemistry and single-molecule bi

通过生物正交化学和单分子双酶切探讨细菌膜中脂多糖的动力学、取向和构象

• 批准号: 2272649
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2272649
• 关键词: Probing; dynamics; orientation; conformation; lipopolysaccharide

The outer membrane (OM) of Gram-negative bacteria forms a significant barrier that helps to protect bacteria from their environment and increases resistance to several antibiotics. The OM is an asymmetric bilayer composed of an inner leaflet of phospholipid and an outer leaflet of lipopolysaccharide (LPS) with OM proteins (OMPs) spanning the bilayer. Recently, we demonstrated that OMPs are turned over via a method termed binary partitioning. New OMPs were shown to be inserted preferentially at the mid-cell with old OMPs pushed to the poles during cell elongation, facilitating fast adaptation to a new environmental niche at the population-level.The spatio-temporal dynamics of LPS in the OM have remained a controversial topic since the introduction of the fluid mosaic model in the 1970s. We propose to finally unequivocally establish the lateral diffusion properties of LPS, explore its potential co-localisation with OMPs, and determine the conformation and orientation of its polysaccharide head group relative to the extracellular surface. This project is wholly reliant upon an interdisciplinary collaboration across the fields of glycoscience, bioorthogonal chemistry and single-molecule microbial biophysics.Using an interdisciplinary approach combining bioorthogonal click-labelling of glycans and single-molecule fluorescence microscopy, we aim to (1) explore the underpinning mechanism governing the spatio-temporal dynamics of LPS through chemical bioconjugation of the LPS insertion machinery in the OM, and simultaneous click-labelling of Kdo sugars in LPS, for fluorescence co-localisation microscopy; and (2) probe the conformation of LPS in the membrane through the dual metabolic labelling of both glycans and acetate groups, and single-molecule FRET experiments.

革兰氏阴性细菌的外膜(OM)形成了一个重要的屏障，有助于保护细菌免受环境的影响，并增加了对几种抗生素的耐药性。OM是一个不对称的双层，由磷脂内叶和脂多糖(LPS)外叶组成，OM蛋白(OMP)横跨该双层。最近，我们证明了OMP是通过一种称为二进制划分的方法来翻转的。在细胞伸长过程中，新的OMP优先插入到细胞中部，旧的OMP被推到两极，有助于在种群水平上快速适应新的环境生态位。自20世纪70年代引入流体镶嵌模型以来，OM中内毒素的时空动态一直是一个有争议的话题。我们建议最终明确地确定内毒素的侧向扩散特性，探索其与OMPS的潜在共定位，并确定其多糖头基相对于细胞外表面的构象和取向。该项目完全依赖于跨糖科学、生物正交化学和单分子微生物生物物理学领域的跨学科合作。我们旨在(1)通过在OM中的脂多糖插入机制的化学生物连接以及在荧光共定位显微镜下同时点击标记脂多糖中的Kdo糖来探索调控脂多糖的时空动力学的基础机制；以及(2)通过糖和醋酸基的双重代谢标记以及单分子FRET实验来探索脂多糖在膜中的构象。