Date of Award

12-20-2024

Date Published

January 2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Davoud Mozhdehi

Keywords

Intrinsically disordered protein;Lipidation;Liquid-liquid phase separation;Post-translational modification

Subject Categories

Chemistry | Physical Sciences and Mathematics

Abstract

Liquid-liquid phase separation (LLPS) is a fascinating biological phenomenon that enables cells to form membraneless organelles, creating specialized compartments for orchestrating complex biochemical reactions. While significant progress has been made in deciphering the sequence determinants underlying phase separation, precisely modulating these features remains a challenge. Inspired by biology, post-translational modifications (PTMs) offer a promising approach to modulate the protein condensates formation and their properties in real-time, without altering the sequences. Despite the identification of over 300 types of PTMs, most research has predominantly focused on charge-altering modifications. The effects of non-charged PTMs, such as lipidation, on LLPS remain poorly understood. This thesis explores how variations in lipidation and operating parameters—such as lipid length, site of modification, and process conditions—can be leveraged to fine-tune the formation, structure and properties of phase-separated condensates. First, we examined how physicochemical properties of lipid influences the formation and kinetic stability of aqueous two-phase system (ATPS) formed by fatty acid-modified elastin-like polypeptides (FAMEs). By systematically varying lipid lengths from C2 to C16 while maintaining consistent polypeptide chemistry, we elucidated the impact of lipid characteristics on the behavior of droplets formed by proteins exhibiting lower critical solution temperature (LCST) behavior. Our findings revealed that the phase separation temperature decreases in a sigmoidal manner with increasing lipid length, highlighting a remarkable sensitivity of temperature-dependent interactions to lipid chain length. These findings enhance our theoretical understanding of protein-lipid interactions and provide a foundation for the rational design of lipidated proteins with precise control over their phase separation. Second, we explored how lipidation and the sequence context of lipidation sites affect the phase behavior and condensates properties of resilin-like polypeptides (RLPs), which exhibit upper critical solution temperature (UCST) transitions. We demonstrated that lipidation, in synergy with the sequence of the lipidation site, dictates the thermodynamic tendency for phase separation and the resulting condensate properties. These findings highlight the importance of "sequence context" in modulating the properties of lipidated protein condensates, indicating that changes in lipidation sequences could be strategically employed to refine the effects of PTMs. Finally, we investigated how temperature-triggered LLPS of post-translationally lipidated proteins can be harnessed to direct the assembly of genetically encoded amphiphilic proteins following tailored thermal pathways. A hybrid protein-based amphiphile with two distinct temperature-responsive domains was designed to study the effects of thermal processing rates, such as quenching and annealing. Through this approach, we successfully synthesized nanomaterials with complex structures that are not thermodynamically favored. Our strategy opens new possibilities for programing the assembly and expanding the structural complexity of protein-based materials. Collectively, these studies deepen our understanding of the role of molecular syntax and operating parameters in governing LLPS, offering new insights into the regulation of disordered protein systems by post-translational modifications. These findings will also contribute to the rational design of biomaterials with precisely controlled phase behavior for biomedical applications.

Access

Open Access

Included in

Chemistry Commons

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