Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Studying the dynamic behavior of large proteins is challenging due to limitations in spectroscopic methods requiring specific probe attachment.
• Amber codon suppression is an emerging technique for incorporating biophysical probes, but its application to very large proteins (MDa) was previously unreported.

Purpose of the Study:

• To investigate the feasibility of using amber codon suppression for site-specific genetic code expansion and fluorescent labeling of a large (540 kDa) homodimeric fatty acid synthase type I.
• To establish a rapid screening method for optimizing suppression conditions for large protein labeling.

Main Methods:

• Utilized a microplate-based reporter assay with a GFP fusion protein to screen for efficient amber codon suppression conditions.
• Applied optimized conditions to express and fluorescently label full-length fatty acid synthase constructs.
• Performed biophysical characterization including HPLC, activity assays, and fluorescence spectroscopy on labeled proteins.

Main Results:

• Successfully demonstrated the introduction of non-canonical amino acids into a 540 kDa fatty acid synthase via amber codon suppression.
• Developed and validated a low-complexity reporter assay for rapid screening of suppression efficiency.
• Generated fluorescently labeled fatty acid synthase for initial biophysical analysis.

Conclusions:

• Amber codon suppression is feasible for site-specific labeling of large, complex multidomain proteins like fatty acid synthase.
• This approach facilitates biophysical studies of protein dynamics and conformational variability.
• Enables potential engineering of molecular machines for improved catalytic function.