Effect of ionic residue sequence on polyampholyte interactions, molecular recognition and phase separation

该项目的总体目标是了解带电两性大分子中的序列编码信息如何实现分子识别并驱动多个非混合相分离液相的形成。液-液相分离的大分子以高度带电的内在无序蛋白结构域的形式存在于不同的生物实体中，如蜘蛛丝纤维的前体、贻贝胶或无膜胞内细胞器。因此，这种凝聚相表现出一系列令人印象深刻的功能，例如，生物活性分子的分配，坚韧纤维的合成或特定的粘附。这些功能是由于定义良好的相互作用和带电序列的分子识别，这可能是由大分子链上带电残基的模式控制的。可以假设，对于带电序列和下游功能（如液-液相分离）之间的分子识别，确实需要对沿链的许多电荷-电荷相互作用进行精细控制。随着序列控制聚合物的出现，这种控制可以实现，并且能够用合成聚合物模拟这些功能将是有趣的。然而，关于带电聚合物如何利用多价离子相互作用来驱动相分离的更多知识必须首先获得。为了实现这一目标，将使用高通量筛选技术、软胶体探针的定量粘附测量以及通过AFM的单分子力谱来破译序列定义的两性水解肽的分子识别模式（A部分：相互作用）。这些定量相互作用研究将结合使用荧光技术和拉曼光谱的液液相分离研究来确定凝聚相的组成和结构（B部分：相分离）。具体目标是通过结合A部分中确定的具有不同自相互作用的不同序列来实现多个非混合分离相。为了更接近地模拟天然内在无序蛋白质的相分离特性，将制备由序列定义肽组成的高分子量结构。在这里，我们提出了针对这种高分子量体系的聚合物模拟反应，这些反应很容易获得，同时保持了带电残基的序列定义。总的来说，该项目将使人们对序列在多两性之间的相互作用和识别、它们的相行为、子相的形成等方面的作用有更广泛的了解，这将超越最先进的技术。这些新的见解可能会激发一类新的材料，它们模仿一种有趣的蛋白质特性：进行功能性相分离的能力。

The overall aim of this project is to understand how sequence-encoded information in charged ampholytic macromolecules achieves molecular recognition and drives the formation of multiple non-mixing phase-separated liquid phases. Liquid-liquid phase-separated macromolecules are present in form of highly charged intrinsically disordered protein domains in vastly different biological entities such as precursors of spider silk fibers, mussel glue or membraneless intracellular organelles. Therefore, such coacervate phases exhibit an impressive array of functions, for example, partitioning of bioactive molecules, synthesis of tough fibers, or specific adhesion. These functions are due to well-defined interactions and molecular recognition of the charged sequences, which is likely controlled by the patterning of charged residues along the macromolecule chain. It can be assumed that fine control over many charge-charge interactions along a chain is indeed required for such molecular recognition among charged sequences and downstream functions, like liquid-liquid phase separation. With the advent of sequence-controlled polymers, such control can be achieved and it would be intriguing to be able to emulate these functions with synthetic polymers. However, more knowledge on how charged polymers take advantage of multivalent ionic interactions to drive phase separation must first be acquired. To achieve this goal, high-throughput screening techniques, quantitative adhesion measurements with soft colloidal probes, and single molecule force spectroscopy via AFM will be used to decipher molecular recognition modes on sequence-defined ampholytic peptides (part A: interactions). These quantitative interaction studies will be combined with liquid-liquid phase separation studies using fluorescent techniques and Raman spectroscopy to determine the composition and structure of the coacervate phases (part B: phase separation). A specific aim is to achieve multiple non-mixing separated phases by combining different sequences with varying self-interactions as determined in part A. In order to more closely mimic the phase separation properties of natural intrinsically disordered proteins, high-molecular-weight structures composed of sequence defined peptides will be prepared. Here, we propose polymer analogue reactions toward such high-molecular-weight systems, which are readily accessible while the sequence defined presentation of charged residues is maintained. Overall, this project will enable a broad understanding of the role of sequence on the mutual interactions and recognition among polyampholytes, their phase behavior, the formation of subphases, etc. which leads beyond the state-of-the-art. These new insights may inspire a new class of materials that mimic an intriguing protein property: the ability to undergo functional phase separation.