title: Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.
abstract: 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 re-action 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.
authors: Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan; Department of Structural Molecular Science, The Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
authors: Yuji
authors: Y
authors: Furutani
authors: Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan; Department of Structural Molecular Science, The Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
authors: Tetsunari
authors: T
authors: Kimura
authors: UNISOKU Co., Ltd., 2-4-3 Kasugano, Hirakata, Osaka 573-0131, Japan.
authors: Kido
authors: K
authors: Okamoto
keywords: attenuated total reflectance; halorhodopsin; ion–protein interaction; membrane protein; stopped-flow technique