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

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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.