Genetically Encoded Lipidation to Manipulate Structure, Assembly, and Phase Behavior of Proteins

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Nearly one in five human proteins are post-translationally lipidated, and while the critical role of post- translational modification in regulating different facets of cell biology (e.g., signaling, membrane localization, etc.) has been well established, many mechanistic questions remain unanswered. These include the effects of lipidation on the energetics, conformations, and function of lipidated proteins (LP) — and on human diseases. Advancing our understanding of protein lipidation at the biophysical level and elucidating the sequence– structure–function rules in various biological milieus, require study of the changes in protein structure and conformation as the physicochemistry of lipid, lipidation site, and proteins are systematically modified. However, such efforts have been stymied by the challenging and laborious methods to synthesize lipid- modified proteins. To advance understanding of protein lipidation, my program will genetically engineer prokaryotes to incorporate a diverse set of lipids into proteins, enabling the rapid generation of comprehensive libraries of model LPs with broad physicochemical diversity. The overarching hypothesis of this program is that the biophysical consequences of protein lipidation is governed by a “molecular syntax”, which is based on the interplay between the physicochemistry of the lipid, protein, and the lipidation site that (de)stabilizes folding or assembly of intermediates via (non)native interactions between lipid, protein sidechains, and the aqueous milieu. To test this hypothesis, diverse and complementary biophysical and soft-matter characterization techniques will be used to (1) study the tertiary structure and quaternary organization of model globular and disordered LPs across three distinct structural hierarchies—single protein chains, lipid-driven supramolecular assemblies, and liquid-liquid phase separation-driven higher-order assemblies (condensates); and (2) quantify the contribution of these structures and the LP’s physicochemistry to encoded functional/material properties such as biomolecular switching, viscoelasticity, and contact mechanics. By establishing platforms to engineer sequence-defined LPs and revealing a rigorous, biophysically rooted molecular syntax underlying their structure and energetics, this research program will substantially broaden the design space and functional landscape of biomolecules beyond protein’s amino acid-based motifs. Ultimately, this program will enable a better understanding of the role of LPs in diverse biological mechanisms in health and disease, and the development of materials and therapeutics with complex structural and functional properties whose capabilities rival natural biosystems for wide applications in nanomedicine and biotechnology.