Development of New Structural Characterisation Tools for Extracellular Matrix

该项目将开发新的结构表征工具，以研究细胞外基质的分子结构以及它如何被非酶化学和细胞活性改变。这将包括在动态核极化（DNP）NMR中开发光谱编辑工具，以便能够从完整的离体基质样品中提取细胞外基质特定区域的NMR光谱。例如，我们将利用关键细胞粘附蛋白通过金属介导的结合与细胞外基质接触的事实，其中天然金属离子可以被顺磁性离子取代，例如在整联蛋白结合位点中Ca2+可以被Co2+取代。顺磁性离子将在其局部淬灭DNP效应，导致DNP NMR光谱中不存在来自整联蛋白结合位点的NMR信号。从含有抗磁性金属离子而不是顺磁性金属离子的样品上收集的光谱中减去该顺磁性编辑的光谱，得到整联蛋白结合位点的NMR光谱。在该项目的第二部分，我们将开发测量细胞外基质在机械应力下的NMR光谱的方法，目的是跟踪该过程中分子构象的变化。总的来说，她的项目将提供新的工具，使人们能够对与衰老和退行性疾病高度相关的生物过程进行新的洞察。

The project will develop new structural characterisation tools to study the molecular structure of extracellular matrix and how it is altered by non-enzymatic chemistry and cellular activity. This will include developing spectral editing tools in Dynamic Nuclear Polarisation (DNP) NMR to be able to extract NMR spectra of specific regions of the extracellular matrix from samples of intact, ex vivo matrix. For instance, we will utilise the fact that key cell adhesion proteins contact the extracellular matrix through metal-mediated binding, where the native metal ion can be replaced by a paramagnetic ion, e.g. Ca2+ can be replaced by Co2+ in integrin binding sites. Paramagnetic ions will quench the DNP effect in their locality, resulting in a DNP NMR spectrum in which NMR signals from the integrin binding site are absent. Subtraction of this paramagnetic-edited spectrum from one collected on the sample containing diamagnetic metal ions instead of paramagnetic gives an NMR spectrum of the integrin binding sites. In a second part of the project we will develop methods to measure NMR spectra on extracellular matrix whilst the matrix is under mechanical stress, with the aim of following changes in molecular conformation during that process.Overall, her project will deliver new tools to enable novel insight into biological processes highly relevant to ageing and degenerative diseases.