Photochemistry of Receptor-Bound Flavin Resolved in Living Human Cells by Infrared Spectroscopy

Area of Science:

• Biophysics
• Spectroscopy
• Cell Biology

Background:

• The cellular environment influences protein structure and function.
• Studying proteins within living cells is crucial for understanding their native mechanisms.

Purpose of the Study:

• Introduce in-cell infrared difference spectroscopy (ICIRD) for studying soluble receptors in living human cells.
• Investigate the in-cell photochemical mechanisms of plant cryptochrome and aureochrome1a (LOV) proteins.
• Assess the impact of the cellular environment on LOV protein structure and hydrogen bonding.

Main Methods:

• Utilized attenuated total reflection with in-cell infrared difference spectroscopy (ICIRD).
• Cultivated and transfected human embryonic kidney cells directly on an internal reflection element within a spectrometer.
• Monitored cell viability and growth in situ using infrared spectroscopy.

Main Results:

• Resolved photoreactions of oxidized flavin to flavin neutral radical in cryptochrome and flavin-cysteine adduct in LOV within living eukaryotic cells.
• Confirmed photochemical mechanisms of cryptochrome and LOV proteins inside mammalian cells.
• Observed a significant upshift in LOV protein carbonyl stretching modes, indicating cellular impact on flavin structure and hydrogen bonding.

Conclusions:

• ICIRD is a noninvasive, label-free method for studying soluble photoactivatable receptors in mammalian cells.
• The eukaryotic cellular environment significantly impacts the hydrogen bonding network and structure of flavin in LOV proteins.
• Findings necessitate consideration of cellular effects in the physiology and optogenetic applications of LOV proteins.