Home › Analytical Chem › Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Analytical Chem JoVE (Open Access) Citable · DOI

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

DOI: 10.3791/51622-v
What you'll learn
  • ✓ Set up attenuated total reflection and transmission FTIR experiments for photosensitive proteins
  • ✓ Synchronize exciting laser with step-scan FTIR instrument for time-resolved measurements
  • ✓ Interpret proton transfer and protein conformational changes on microsecond-millisecond timescales
  • ✓ Collect and analyze time-resolved step-scan FTIR data from bacteriorhodopsin and channelrhodopsin-2
Protocol

Biopharma Insights Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Difficulty
advanced
Total time
~4–6 hours per sample (instrument setup, alignment, data acquisition)

Steps

1
Prepare attenuated total reflection experiments on bacteriorhodopsin

Configure ATR-FTIR setup for bacteriorhodopsin samples. Position crystal, apply sample, and optimize optical coupling for infrared transmission.

▶ 02:13
2
Perform transmission experiments on detergent-solubilized channelrhodopsin-2

Set up transmission FTIR geometry for solubilized channelrhodopsin-2 in detergent micelles. Optimize sample cell path length and baseline acquisition.

▶ 03:48
3
Adjust and synchronize the exciting laser

Align the pump laser to the sample, optimize beam overlap with FTIR probe beam, and synchronize laser triggering with step-scan timing.

▶ 05:02
4
Configure time-resolved step-scan FTIR settings

Define temporal windows, step intervals, and laser pulse delays. Program spectrometer parameters for microsecond-to-millisecond time resolution.

▶ 05:55
5
Collect and interpret time-resolved step-scan FTIR results

Acquire time-resolved spectra following laser excitation. Extract protonation state and backbone conformation dynamics from infrared absorption changes.

▶ 07:48
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