title: Proton transfer and protein conformation dynamics in photosensitive proteins by time-resolved step-scan Fourier-transform infrared spectroscopy.
abstract: Monitoring 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.
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin; jheberle@physik.fu-berlin.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D055668:Photochemical Processes; D011487:Protein Conformation / Q000528:radiation effects; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D011506:Proteins; D011522:Protons; D001436:Bacteriorhodopsins; D012243:Rhodopsin