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

近五分之一的人类蛋白质是翻译后脂化的，虽然后翻译后脂肪的关键作用是 调节细胞生物学的不同方面的翻译修饰(例如，信号、膜定位， 等)尽管已经得到了很好的证实，但许多机械性的问题仍然没有得到回答。这些影响包括 脂化作用对脂化蛋白质(LP)的能量、构象和功能的影响--以及对人类疾病的影响。 在生物物理水平上推进我们对蛋白质脂肪作用的理解，并阐明其序列-- 在各种生物环境中的结构-功能规律，需要研究蛋白质结构和功能的变化 作为脂类、脂化部位和蛋白质的物理化学的构象被系统地修饰。 然而，由于合成脂质的方法具有挑战性且费时费力，这些努力受到了阻碍。 修饰的蛋白质。为了促进对蛋白质脂肪作用的理解，我的程序将通过基因工程 原核生物将一组不同的脂类结合到蛋白质中，使快速生成全面的 具有广泛物理化学多样性的模型LP文库。这一计划的首要假设是 蛋白质脂肪作用的生物物理后果受一种“分子语法”支配，这种语法是基于 脂类、蛋白质的物理化学与(去)稳定折叠或 通过脂类、蛋白质侧链和水分子之间的(非)天然相互作用组装中间体 周围的环境。为了验证这一假设，多样化和互补性的生物物理和软物质表征 技术将被用来(1)研究模型球状和 在三个不同的结构层次上无序的LP-单蛋白链，脂质驱动的超分子 组件和液-液相分离驱动的高阶组件(凝析油)；以及(2)量化 这些结构和LP的物理化学对编码的功能/材料性质的贡献 如生物分子开关、粘弹性和接触力学。通过建立工程设计平台 序列定义的LP，并揭示了其背后严格的、生物物理基础的分子语法 结构和能量学，这一研究计划将大大拓宽设计空间和功能 超越蛋白质的氨基酸基序的生物分子景观。最终，该计划将使 更好地了解脂蛋白在健康和疾病的不同生物机制中的作用，以及 具有复杂结构和功能特性的材料和治疗剂的开发 在纳米医学和生物技术中广泛应用的与之竞争的天然生物系统。

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.