Engineering a periplasmic binding protein for amino acid sensors with improved binding properties

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Periplasmic binding proteins (PBPs) are crucial for solute uptake in bacteria and archaea.
• Engineering PBPs offers a pathway to create novel biosensors for specific molecules.

Purpose of the Study:

• To engineer a leucine-binding PBP into a sensitive and specific biosensor for l-leucine detection.
• To utilize a Förster resonance energy transfer (FRET) system with a fluorescent unnatural amino acid and yellow fluorescent protein (YFP).

Main Methods:

• Genetic incorporation of a fluorescent unnatural amino acid (CouA) as a FRET donor.
• Fusion of YFP as a FRET acceptor to the PBP N-terminus.
• Protein engineering to enhance binding affinity, specificity, and sensitivity.

Main Results:

• Engineered PBP showed a 2.5-fold increase in FRET ratio upon l-leucine binding at position 178.
• The sensor exhibited high specificity, with minimal response to other natural amino acids and d-leucine.
• Further modifications yielded a 14-fold increase in sensitivity for l-leucine and recognition of l-methionine.

Conclusions:

• The FRET-based sensor design strategy successfully engineered a natural receptor for improved binding affinity and specificity.
• The developed sensor can quantify l-leucine in biological samples and determine the optical purity of amino acids.
• This approach is applicable to engineering other receptors for sensing diverse biochemically relevant molecules.