Switchable elastin-like polypeptides that respond to chemical inducers of dimerization

Area of Science:

• Biotechnology
• Protein Engineering
• Materials Science

Background:

• Elastin-like polypeptides (ELPs) are protein polymers known for reversible phase separation triggered by environmental factors like temperature and pressure.
• Current limitations exist in engineering ELPs to respond to specific molecular substrates, hindering their application in advanced biomaterials and therapeutics.
• Developing strategies for substrate-specific ELP response is crucial for unlocking their full potential as biosensors, diagnostic agents, and targeted therapies.

Purpose of the Study:

• To develop a novel strategy for engineering ELP fusion proteins that respond to specific molecular substrates.
• To demonstrate that ELP phase separation can be controllably induced by chemical inducers of dimerization (CID).
• To establish a method for designing ELP-based systems with tunable, substrate-specific phase separation properties.

Main Methods:

• Constructs were designed by fusing an immunophilin, human FK-506 binding protein 12 (FKBP), to an ELP.
• The phase separation behavior of these fusion proteins was evaluated in response to varying concentrations of CID.
• ELP molecular weight was modulated to fine-tune the phase separation for isothermal response at physiological conditions (37 °C, physiological ionic strength).
• An empirical mathematical model was used to analyze the relationship between transition temperature and binding constants.

Main Results:

• ELP fusion proteins demonstrated reversible phase separation triggered by stoichiometric binding of CID, leading to homodimerization.
• Phase separation was reversible upon excess CID saturation or competitive binding of small molecules to FKBP.
• The molecular weight of the ELP component allowed for tuning the phase separation to occur isothermally at 37 °C and physiological ionic strength.
• This study presents the first demonstration of reversible ELP switching controlled by specific dimerization.

Conclusions:

• Chemical inducers of dimerization (CID) provide a robust strategy to engineer ELP fusion proteins for specific molecular responses.
• This approach enables the creation of tunable, reversible ELP-based systems for applications in biosensing, diagnostics, and targeted therapies.
• The simplicity of this method suggests broad utility for designing ELP fusion proteins responsive to specific dimeric biological entities.