Genetic Code Expansion for Site-Specific Encoding of a Switchable, Intrinsic Fluorophore-Quencher Pair to Monitor...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Site-specific protein modification in native structures is crucial for biological studies.
• Existing methods often require post-translational labeling, limiting experimental flexibility.

Purpose of the Study:

• To present a genetic code expansion strategy for integrating a fluorophore-quencher pair (acridonylalanine and methyltetrazinyl phenylalanine) into proteins.
• To enable internal control experiments by switching off quenching via biorthogonal or photochemical reactions.
• To demonstrate the utility of this method in studying protein dynamics and structure-function relationships.

Main Methods:

• Utilized genetic code expansion in *E. coli* to incorporate two non-canonical amino acids (Acd and Tet).
• Performed mechanistic studies including Stern-Volmer quenching and fluorescence lifetime measurements.
• Applied the dual labeling strategy to calmodulin, RecA, and LexA proteins.

Main Results:

• Successfully demonstrated site-specific dual labeling of proteins with Acd and Tet.
• Established the distance dependence of quenching for the Acd/Tet pair.
• Provided molecular-level insights into the dynamics of calmodulin, RecA, and LexA.

Conclusions:

• The Acd/Tet genetic code expansion strategy offers a powerful tool for studying protein dynamics and structure-function relationships.
• This method facilitates high-throughput drug screening, particularly for antibiotic resistance mechanisms.
• The ability to switch quenching on/off provides convenient internal controls for experiments.