Single Molecule Visualization of Chitin/Chitosan Structures and their Protein Interactions

本项目旨在开发在单分子水平上分析甲壳素/壳聚糖结构及其蛋白质相互作用的工具。甲壳素和壳聚糖是继纤维素之后地球上最丰富的生物分子。甲壳素/壳聚糖多糖是许多生命系统中重要的结构材料和信号分子，由于其低成本和生物相容性，它们具有重要的商业用途。尽管它们的重要性和普遍性，但是，很少有人知道甲壳素/壳聚糖的结构，以及它们如何转化为甲壳素/壳聚糖的性能，由于在分析它们的结构，通过合奏平均分析技术的困难。鉴于甲壳质是乙酰化单糖的长的线性链，而壳聚糖是乙酰化和非乙酰化单糖的共聚物，总体平均方法不能提供关于沿沿着单独甲壳质/壳聚糖链存在的乙酰化模式/基序的信息。通过系综平均方法分析这些多糖的困难进一步加剧了它们在单个分子中的广泛结构变化。揭示甲壳素/壳聚糖结构和相互作用的挑战需要直接的单分子方法。该项目通过使用甲壳素/壳聚糖的直接成像来面对这一挑战，以揭示它们在单分子水平上的结构和相互作用。甲壳素/壳聚糖电喷雾作为离子在气相中，随后降落完整的表面和成像单独通过扫描隧道显微镜在亚分子分辨率。通过在纳米尺度上对单个甲壳素/壳聚糖链进行成像，我们的目标是揭示乙酰化序列、形状/折叠以及单个甲壳素/壳聚糖链的相互作用。具体而言，将研究三个系统，即（1）甲壳素/壳聚糖寡糖，（2）甲壳素/壳聚糖多糖，和（3）蛋白质-甲壳素/壳聚糖加合物。甲壳质/壳聚糖的直接纳米级成像预计将揭示促进其与启动或调节生物功能的不同蛋白质结合的结构基序，以及引起甲壳质/壳聚糖材料丰富物理性质的基序。在单分子水平上理解甲壳素/壳聚糖的结构和相互作用将为理解甲壳素/壳聚糖的不同性质提供分子基础，从而有助于开发基于甲壳素和壳聚糖的新的治疗策略和功能材料。

This project aims to develop tools to analyze structures of chitin/chitosan and their protein interactions at single molecule level. Chitin and chitosan are the most abundant biomolecules on Earth after cellulose. Chitin/chitosan polysaccharides are important structural materials and signaling molecules in many living systems, and they have vital commercial uses due to their low cost and biocompatibility. Despite their importance and ubiquity, however, little is known about chitin/chitosan structures and how they translate to chitin/chitosan properties, due to difficulties in analyzing their structures by ensemble averaged analytical techniques. Given that chitin is a long, linear chain of acetylated monosaccharides while chitosan is a copolymer of acetylated and non-acetylated monosaccharides, ensemble averaged methods are unable to provide information on acetylation pattern/motif present along individual chitin/chitosan chains. Difficulties in analyzing these polysaccharides by ensemble averaged methods are further exacerbated by their extensive structural variations among individual molecules. The challenge of unveiling chitin/chitosan structures and interactions calls for a direct, single molecule approach. This project confronts this challenge by using direct imaging of chitin/chitosan to unveil their structures and interactions at single molecule level. Chitin/chitosan are electrosprayed as ions in gas phase which are subsequently landed intact at a surface and imaged individually by scanning tunnelling microscopy at submolecular resolution. By imaging single chitin/chitosan chains at the nanoscale, we aim to reveal the acetylation sequence, the shape/folding, and the interactions of individual chitin/chitosan chains. Specifically, three systems will be investigated, namely (1) chitin/chitosan oligosaccharides, (2) chitin/chitosan polysaccharides, and (3) protein – chitin/chitosan adducts. Direct nanoscale imaging of chitin/chitosan is anticipated to unveil structural motifs that facilitate their binding to diverse proteins which initiate or regulate biological functions, as well as motifs that give rise to rich physical properties of chitin/chitosan materials. Understanding chitin/chitosan structures and interactions at single molecule level will provide the molecular basis to understand the diverse chitin/chitosan properties, thereby contributing towards the development of new therapeutic strategies and functional materials based on chitin and chitosan.