Covalent Drug Binding in Live Cells Monitored by Mid-Infrared Quantum Cascade Laser Spectroscopy: Photoactive Yellow...

Area of Science:

• Biochemistry
• Spectroscopy
• Molecular Biology

Background:

• Genetically encoded vibrational probes, like nitriles, offer sensitive detection of molecular interactions in live cells.
• Measuring nitrile vibrational probes in live cells is challenging due to low signal-to-noise ratios.
• Quantum cascade laser (QCL) technology can potentially enhance sensitivity for these measurements.

Purpose of the Study:

• To design and implement a highly sensitive QCL-based spectrometer for detecting drug-target interactions in live cells.
• To demonstrate the system's capability in monitoring small-molecule binding within *Escherichia coli*.
• To investigate the interaction between para-Coumaric acid (pCA) and nitrile-incorporated photoactive yellow protein (PYP) as a model system.

Main Methods:

• Development of a double-beam QCL-based transmission infrared (IR) spectrometer with balanced detection.
• Utilizing nitrile vibrational probes embedded in proteins within live *Escherichia coli*.
• Employing high-level molecular dynamics simulations with the AMOEBA force field for spectral shift analysis.

Main Results:

• The QCL spectrometer significantly enhanced sensitivity for nitrile vibrational probes in live bacteria compared to FTIR.
• Detected small-molecule binding, specifically pCA to nitrile-PYP, directly within *E. coli*.
• Observed large spectral shifts (up to 15 cm^-1) in nitriles upon drug binding, correlating with purified protein studies.

Conclusions:

• The QCL spectrometer provides enhanced sensitivity for monitoring drug-protein interactions in a cellular context.
• This methodology advances drug development and biochemical research by enabling in-cell interaction studies.
• Observed spectral shifts are attributed to altered hydrogen-bonding environments, validated by molecular dynamics simulations.