Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode

Furutani, Yuji; Kimura, Tetsunari; Okamoto, Kido

doi:10.2142/biophysics.9.123

Cited by 12 publications

(21 citation statements)

References 20 publications

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“…Time-resolved step-scan spectroscopy is only straightforwardly applicable to proteins showing reversible reactions that can be triggered reproducibly by light (but see progress in coupling step-scan with rapid buffer exchange) 71 . In step-scan the reaction needs to be reproducible for at least ~500x, the minimum number to complete an interferogram covering the 1,800-850 cm -1 region at 8 cm -1 resolution.…”

Section: Discussionmentioning

confidence: 99%

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

Lórenz-Fonfrı́a

Heberle

2014

JoVE

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Citation: Lórenz-Fonfría, V.A., Heberle, J. Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Stepscan Fourier-transform Infrared Spectroscopy. J. Vis. Exp. (88), e51622, doi:10.3791/51622 (2014). AbstractMonitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~10 2 -10 3 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10 -4 , sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/ or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

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Section: Discussionmentioning

confidence: 99%

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

Lórenz-Fonfrı́a

Heberle

2014

JoVE

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“…Various kinds of ion channels and pumps are potential targets for this method. Recently, a rapid-buffer exchange method utilizing syringe pumps driven by pressured gas has been developed which enables the rapid exchange of buffer solutions on ATR crystal (typically within 10-30 ms) (Furutani et al 2013;Shirai et al 2014). This technique may be utilized to detect transient conformational changes involving ion-binding processes in proteins.…”

Section: Structural Difference Of the Selectivity Filter Interacting mentioning

confidence: 99%

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

Furutani

2017

Biophys Rev

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An understanding of ion-protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion-protein interactions in the KcsA selectivity filter. A band at 1680 cm in the amide I region would be a marker band for the ion occupancy of K, Rb, and Cs in the filter. The band at 1627 cm observed in both Na and Li conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K ions provides quantitative information on the ion affinity of the selectivity filter.

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“…Cysteine-scanning is applicable for photoactive protein, for which difference FTIR spectra is easily measured. Recently rapid buffer exchange system using attenuated total reflection cell is developed for FTIR 22 . By combination of these techniques, cysteine-scanning is applicable for a variety of proteins.…”

Section: Discussionmentioning

confidence: 99%

Mapping of the local environmental changes in proteins by cysteine scanning

et al. 2014

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Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.

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Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode

Cited by 12 publications

References 20 publications

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

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

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

Mapping of the local environmental changes in proteins by cysteine scanning

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