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

Abstract

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.

pH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin

“…The PMs were washed by centrifugation and resuspended in 3 mM MES and 2 mM NaCl at pH 6.3. Around 10-20 μL of this solution was placed on top of a BaF 2 window and dried under ambient humidity, followed by rehydration in an atmosphere of 99% relative humidity, as previously described (12,88). Equivalent films were prepared using MESd 12 (Cambridge Isotope Laboratories, Inc.) as a buffer, a molecule where all of the hydrogens of MES unchangeable in water are substituted by deuterium atoms.…”

“…Step-Scan FTIR Spectroscopy. We performed time-resolved step-scan FTIR spectroscopy essentially as described before (12). Light-adapted BR films containing either the buffer MES or MESd 12 , were excited by a 10-ns laser pulse (532 nm, 2 mJ/cm 2 , 10 Hz) and time-resolved spectra were obtained at 6.25-μs temporal and 8-cm −1 spectral resolution.…”

“…Protonation changes in the surrounding aqueous medium have been mostly probed in the visible range using pH-indicating dyes. These experiments require diluted and very weakly buffered protein solutions (8)(9)(10)(11), in contrast to the well-buffered and highly concentrated samples used in IR spectroscopy (4,12). It was soon realized that under low concentration of mobile buffers the released protons remain temporally trapped along the membrane surface, being detected by dye molecules in the bulk phase with a delay of ∼0.5-1 ms (8,9,13,14).…”

Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin

“…FTIR transmission difference spectroscopy was performed at 22 1C using Bruker Vertex80v spectrometer, 51 in three different regimes (steady-state, rapid scan, and step-scan). Broadband time-resolved FTIR experiments with ms time resolution were performed by applying the step-scan technique as described previously.…”

Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s

“…Rehydrated film samples of bR in the native purple membrane (PM) were prepared as described in [35,36]. Briefly, ~5 µl of a stock solution of PM (~5 mg/ml in 3 mM KCl and 3 mM potassium phosphate solution, pH 6.5) were deposited on a CaF 2 window and dried to form a film with a diameter of 3 mm.…”