Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode
-
- Furutani Yuji
- Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science Department of Structural Molecular Science, The Graduate University for Advanced Studies (SOKENDAI)
-
- Kimura Tetsunari
- Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science Department of Structural Molecular Science, The Graduate University for Advanced Studies (SOKENDAI)
-
- Okamoto Kido
- UNISOKU Co., Ltd.
書誌事項
- 公開日
- 2013
- 資源種別
- journal article
- DOI
-
- 10.2142/biophysics.9.123
- 公開者
- 日本生物物理学会
説明
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. Time-resolved 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; 2.5 ms), we confirmed the completion of the buffer-exchange reaction within ~25 ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm-1. We also demonstrated the anion-binding 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.<br>
収録刊行物
-
- BIOPHYSICS
-
BIOPHYSICS 9 (0), 123-129, 2013
日本生物物理学会
