Engineered solubility tag for solution NMR of proteins

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Low protein solubility impedes large-scale expression, purification, and biophysical characterization.
• Aggregation-prone proteins, such as the CARD domain of ASC, present significant challenges in research.
• Standard biophysical techniques like NMR spectroscopy are often limited by poor protein solubility.

Purpose of the Study:

• To develop a general and straightforward strategy for enhancing protein solubility.
• To improve the feasibility of large-scale protein expression, purification, and biophysical measurements.
• To demonstrate the efficacy of the method on the aggregation-prone ASC CARD domain.

Main Methods:

• Conjugating the target protein to a re-engineered, highly soluble 6 kDa protein tag at a solvent-exposed position.
• Utilizing site-directed mutagenesis to introduce a cysteine residue at the self-association interface of the ASC CARD domain.
• Employing NMR spectroscopy to assess the improved biophysical properties of the solubilized protein variant.

Main Results:

• Achieved over 50-fold improvement in ASC CARD solubility under physiological conditions.
• Demonstrated significantly enhanced NMR relaxation parameters for the solubilized ASC CARD variant.
• Confirmed that the tag placement minimally perturbs the target protein's structure and function.

Conclusions:

• The developed protein conjugation strategy offers a versatile approach to overcome solubility limitations.
• This method facilitates advanced biophysical studies, including NMR, on previously intractable proteins.
• The technique is broadly applicable for improving the solubility and handling of diverse proteins for research and development.