Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an...

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• Non-polar surface patches on proteins can decrease solubility and induce aggregation.
• Protein aggregation impacts protein stability and function.
• Understanding factors influencing protein solubility is key for protein engineering.

Purpose of the Study:

• To investigate methods for reversing aggregation caused by non-polar patches in proteins.
• To determine the effect of charged residues on the solubility and stability of proteins with non-polar patches.
• To explore the role of negatively charged residues in mitigating protein aggregation.

Main Methods:

• Engineering a mutant protein (6L) from sulerythrin with an introduced non-polar leucine patch.
• Introducing negatively charged residues (glutamates, aspartates) or positively charged residues (lysines, arginines) near the non-polar patch.
• Assessing protein solubility, aggregation, and alpha-helical content of wild-type and mutant proteins.

Main Results:

• Mutant 6L with a non-polar patch showed reduced solubility and aggregated.
• Introducing three to six negatively charged residues improved solubility but did not fully prevent aggregation in all cases.
• Mutants with six additional negatively charged residues exhibited dimer formation and retained wild-type alpha-helical content.
• Positively charged residues were less effective than negatively charged residues in restoring solubility.

Conclusions:

• Negatively charged residues surrounding a non-polar patch can effectively reverse solubility reduction and aggregation in acidic proteins.
• The type and number of charged residues are critical for restoring protein solubility and stability.
• This study provides insights into protein design principles for enhanced stability and function.