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

近五分之一的人类蛋白质是后脂肪化的，而后脂肪化的关键作用是， 在调节细胞生物学的不同方面的翻译修饰（例如，信号传导，膜定位， 等等）。虽然已经很好地建立了，但许多机械问题仍然没有答案。这些影响包括： 脂化对脂化蛋白质（LP）的能量、构象和功能以及人类疾病的影响。 在生物物理水平上推进我们对蛋白质脂化的理解，并阐明序列- 在各种生物环境中的结构-功能规则，需要研究蛋白质结构的变化， 由于脂质的物理化学性质、脂化位点和蛋白质的构象被系统地改变， 然而，这些努力受到合成脂质的具有挑战性和费力的方法的阻碍。 改性蛋白质为了增进对蛋白质脂化的理解，我的项目将通过基因工程 原核生物将一组不同的脂质整合到蛋白质中，从而能够快速生成全面的 具有广泛物理化学多样性的模型LP文库。这个项目的首要假设是， 蛋白质脂化的生物物理后果由“分子语法”控制， 脂质、蛋白质和脂化位点的物理化学之间的相互作用（去）稳定折叠或 通过脂质、蛋白质侧链和水溶液之间的（非）天然相互作用组装中间体 环境。为了验证这一假设，多样和互补的生物物理和软物质表征 技术将用于（1）研究模型球状和球状的三级结构和四级组织， 无序脂蛋白跨越三个不同的结构层次-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（冷凝物）;以及（2）量化 这些结构和LP的物理化学对编码的功能/材料特性的贡献 例如生物分子转换、粘弹性和接触力学。通过建立平台， 序列定义的脂多糖，并揭示了一个严格的，生物药理学根源的分子语法，其基础 结构和能量，这项研究计划将大大拓宽设计空间和功能 蛋白质氨基酸基序之外的生物分子景观。最终，该计划将使 更好地了解LP在健康和疾病的各种生物机制中的作用， 开发具有复杂结构和功能特性的材料和治疗剂， 在纳米医学和生物技术的广泛应用中与自然生物系统竞争。

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.