Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Timeresolved measurements require a trigger, which can be controlled electronically; therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding reaction of a protein. By using the step-scan mode (time resolution; 2.5ms), we confirmed the completion of the buffer-exchange reaction within ~25ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm-1. We also demonstrated the anionbinding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm-1. It also should be noted that low sample consumption (~1 μg of protein) makes this new method a powerful technique to understand ligand-protein and ion-protein interactions, particularly for membrane proteins. ©2013 THE BIOPHYSICAL SOCIETY OF JAPAN.
CITATION STYLE
Furutani, Y., Kimura, T., & Okamoto, K. (2013). Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode. Biophysics (Japan), 9, 123–129. https://doi.org/10.2142/biophysics.9.123
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