Modulating Phase Behavior in Fatty Acid-Modified Elastin-like Polypeptides (FAMEs): Insights into the Impact of Lipid Length on Thermodynamics and Kinetics of Phase Separation

Abstract

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid−liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous twophase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7−C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein−lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

Lipidation alters the phase-separation of resilin-like polypeptides

“…Importantly, we observed that lipidated RLPs maintained their concentration-dependence in their cloud point temperatures—a finding contrasting with the effects of lipidation on ELPs, where it notably decreases the slope of temperature-composition diagrams. 38,39 Additionally, a notable difference was observed in the Y -intercepts of these lines, which underscores the synergistic interaction between the sequence of the lipidation site and the lipid. Here, the Y -intercept values for AGA -RLP and LSL -RLP were determined to be −6.4 °C (CI: [−7.9, −4.9]) and −5.3 °C (CI: [6.8, −3.8]), respectively.…”