Modulation of protein stability and aggregation properties by surface charge engineering.

Area of Science:

• Biochemistry
• Protein Engineering
• Structural Biology

Background:

• Traditional protein engineering altering surface charges often leads to protein misfolding and structural instability.
• Modulating protein properties via surface charge modification is hindered by these destabilizing effects.

Purpose of the Study:

• To overcome limitations in protein surface charge engineering by co-introducing stabilizing mutations.
• To investigate if protein stability can be modulated independently of activity and folding through surface charge engineering.

Main Methods:

• Designed rational mutations to increase and decrease surface amino acid charges in Green Fluorescent Protein (GFP).
• Co-introduced stabilizing mutations with charge-modifying mutations into GFP.
• Assessed functional activity, folding rates, kinetic stability, and aggregation properties of engineered GFP variants.

Main Results:

• Functionally active GFP variants with altered surface charges (s-GFP(+15-17) and s-GFP(+5-6)) were successfully generated by co-introducing stabilizing mutations.
• Protein stability was modulated independently of activity and folding rate through surface charge engineering.
• Engineered surface charges altered the aggregation properties of GFP.

Conclusions:

• Co-introduction of stabilizing mutations is an effective strategy to maintain protein function during surface charge engineering.
• Protein surface charge engineering offers a viable approach to independently modulate protein stability, activity, and aggregation properties.