biostudies-literature00460Unknown9Furutani YAttenuated 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.Biophysics (Nagoya-shi, Japan)123-9https://www.ebi.ac.uk/biostudies/studies/S-EPMC4629687biostudies-literatureDevelopment of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.PMC4629687Furutani YKimura TOkamoto K46falseDevelopment of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.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.2013-01-01T00:00:00Z20132021-03-06T08:31:17Z2019-03-27T02:01:10ZS-EPMC46296872749355010.2142/biophysics.9.123