Structure-based substitutions for increased solubility of a designed protein.

Area of Science:

• Protein engineering and design
• Biochemistry
• Structural biology

Background:

• Protein solubility is crucial for protein design and engineering applications.
• Consensus ankyrin repeat proteins (ANK) were previously designed as scaffolds.
• The 4ANK protein, while stable, exhibits solubility only under acidic conditions.

Purpose of the Study:

• To improve the solubility of engineered ankyrin repeat proteins at physiological pH.
• To investigate the effect of surface residue modification on protein solubility and stability.
• To enable the use of engineered proteins as scaffolds for interactions at physiological pH.

Main Methods:

• Designed a series of consensus ankyrin repeat proteins (1ANK, 2ANK, 3ANK, 4ANK).
• Modified the surface of 4ANK by substituting hydrophobic leucine residues with positively charged arginine residues.
• Assessed protein solubility across a range of pH values.
• Studied the pH dependence of protein stability.

Main Results:

• Substitution of six surface leucines with arginines in 4ANK significantly increased its solubility at physiological pH.
• The modified 4ANK protein demonstrated enhanced solubility over a broad pH range.
• Stability studies confirmed 4ANK as a highly stable ankyrin repeat protein.
• Leucine to arginine substitutions on 2ANK promoted a transition from a partially folded to a fully folded conformation.

Conclusions:

• Replacing surface-exposed hydrophobic residues with positively charged residues is an effective strategy to enhance protein solubility at physiological pH.
• This approach can improve the utility of engineered proteins as scaffolds for biological applications.
• Protein surface chemistry plays a critical role in determining solubility and folding behavior.