Assembling membraneless organelles from de novo designed proteins

Area of Science:

• Synthetic Biology
• Protein Engineering
• Biophysics

Background:

• De novo protein design has yielded diverse protein structures and complexes.
• A key challenge is applying these designs within living cells for biological intervention.
• Designing self-assembling structures like membraneless organelles is a significant hurdle.

Purpose of the Study:

• To design genetically encoded polypeptides that self-assemble into membraneless organelles in Escherichia coli.
• To demonstrate the utility of these designed organelles for cellular processes and synthetic biology applications.

Main Methods:

• Combined de novo alpha-helical sequences, intrinsically disordered linkers, and client proteins into single-polypeptide constructs.
• Tailored helical region properties to control protein assembly dynamics, shifting from arrested assemblies to dynamic condensates.
• Characterized designed polypeptides in vitro and in vivo using biophysical methods and soft-matter physics principles.

Main Results:

• Successfully designed and characterized polypeptides that form functional membraneless organelles in E. coli.
• Demonstrated control over assembly properties, transitioning from static to dynamic structures.
• Achieved efficient co-compartmentalization of an enzyme pair, significantly enhancing product formation.

Conclusions:

• Genetically encoded polypeptides can be designed to form functional membraneless organelles in bacteria.
• This approach offers a powerful tool for bottom-up construction of cellular microcompartments.
• The designed system enhances enzymatic efficiency by concentrating reaction components, approaching theoretical limits.