A topological refactoring design strategy yields highly stable granulopoietic proteins

Area of Science:

• Biochemistry
• Protein Engineering
• Structural Biology

Background:

• Protein therapeutics often suffer from production, stability, and solubility issues.
• De novo protein design offers a promising strategy to overcome these limitations.

Purpose of the Study:

• To demonstrate the utility of topological refactoring for designing novel granulopoietic proteins.
• To create engineered proteins with improved therapeutic properties compared to native granulocyte-colony stimulating factor (G-CSF).

Main Methods:

• Applied topological refactoring to the G-CSF structure by rearranging its sequence and optimizing for a new fold.
• Designed and tested four novel protein candidates.
• Evaluated protein activity, thermostability, protease resistance, structural accuracy, and in vitro/in vivo efficacy.

Main Results:

• Two out of four designed proteins exhibited nanomolar activity.
• The most active design showed high thermostability, protease resistance, and atomic accuracy to its designed structure.
• Engineered proteins, despite sequence and structural differences from native G-CSF, specifically differentiated human hematopoietic stem cells into neutrophils.
• Demonstrated significant and specific in vivo activity.

Conclusions:

• Topological refactoring is an effective strategy for de novo protein design.
• This approach can yield highly stable and active protein therapeutics.
• The method is broadly applicable to various protein targets independent of sequence or structural context.