Force-induced Conformational Transitions in Single Polysaccharide Molecules by AFM

在生物系统（如葡萄糖）中，单多糖分子由afpyranose环引起的力诱导构象转变是非常重要的。许多具有重要生物学意义的多糖是由吡喃糖环组成的，它们在各种细胞结构（如植物细胞壁或动物组织中的细胞外基质）中处于拉伸应力下。机械应力被认为调节这些复杂弹性系统的组装和生理特性。多糖通过其成分的机械重排来响应这种压力，但其潜在机制尚不清楚。最简单的观点认为多糖是熵弹簧，吡喃糖环结构通常被描绘成无弹性的，并锁定在一个稳定的构象中。单分子原子力显微镜技术的发展使得对这些观点进行批判性的检验成为可能。AFM仪器可以机械地拉伸单个分子，并具有极好的长度和力分辨率。用原子力显微镜对多糖进行拉伸，结果表明多糖不表现为简单的熵弹簧，而表现为屈服现象。这些弹性偏差的来源被确定为吡喃糖环，该环被发现在拉伸时经历构象转变，如椅-船或椅反转。这些强制转变以循序渐进的方式改变了糖苷氧原子的分离，因此影响了多糖链的轮廓长度及其弹性。轴向糖苷键被发现通过充当原子杠杆来驱动这些转变。本建议的长期目标是在原子水平上理解多糖中这些力诱导的构象转变的机制。在第二个资助期间，单分子原子力显微镜技术将与计算化学工具结合，详细检查吡喃糖环中力诱导的构象转变。通过从不同的方向和不同的附着点拉环来探测环的机械性能及其机械构象转变。这些研究将确定每种类型的单体对混合链的复合弹性的贡献，并将对解释许多天然多糖的分子弹性有价值。本提案的实验和理论发现将被整合到开发一种基于原子力显微镜的方法，用于从其独特的力扩展光谱中识别溶液中的单个多糖分子。这种方法将是对碳水化合物研究分析工具库的重要补充。

Force-induced conformational transitions in single polysaccharide molecules by AFMPyranose ring-based molecules are of extraordinary importance to biological systems (e.g.glucose). Many biologically important polysaccharides are composed of pyranose rings and theyare placed under tensile stress in a wide variety of cellular structures such as the cell wall ofplants or the extracellular matrix (ECM) in animal tissues. Mechanical stress is thought toregulate assembly and physiological properties of these complex elastic systems. Thepolysaccharides respond to that stress by mechanical rearrangements of their components butthe underlying mechanism is not well understood. The simplest view assumes thatpolysaccharides are entropic springs and the pyranose ring structure is typically portrayed asinelastic and locked into a stable conformation. The development of single molecule AFMtechniques allowed for critically examining these views. AFM instruments can stretchmechanically single molecules and have superb length and force resolution. Stretching ofpolysaccharides by AFM revealed that they do not behave as simple entropic springs but thatthey display yielding phenomena. The origin of these elastic deviations was pinpointed to thepyranose ring that was found to undergo, upon stretching, conformational transitions such aschair-boat or chair inversion. These forced transitions change, in a step-wise fashion, theseparation of the glycosidic oxygen atoms, and therefore affect the contour length of thepolysaccharide chain and its elasticity. Axial glycosidic bonds were found to drive thosetransitions by acting as atomic levers. The long-term objective of this proposal is to understand,at the atomic level, the mechanism of these force-induced conformational transitions inpolysaccharides. During the second grant period single molecule AFM techniques will becombined with the tools of computational chemistry to examine in detail force-inducedconformational transitions in the pyranose ring. The mechanical properties of the ring and itsmechanical conformational transitions will be probed by pulling on the ring from variousdirections and by different attachment points. These studies will determine the contribution of each type of the monomer to thecomplex elasticity of a mixed chain and will be valuable in interpreting the molecular elasticity ofmany native polysaccharides. The experimental and theoretical findings of this proposal will beintegrated to develop an AFM-based methodology for identifying individual polysaccharidemolecules in solution from their unique force-extension spectra. Such a methodology will be animportant addition to the arsenal of analytical tools for carbohydrate research.