Lecture of Dr. Victor A. Lorenz-Fonfria from the Institute of Molecular Science in Spain

Abstract of lecture:

Time-resolved FT-IR difference spectroscopy of proteins: from principles to applications

Victor A. Lorenz-Fonfria

Institute of Molecular Science (ICMol), Universitat de València, 46980 Paterna, Spain

FTIR spectroscopy is sensitive to molecular vibrations. But when applied to proteins, FTIR spectroscopy suffers from a strong background absorbance from the solvent and from band crowding due to the many overlapping vibrations present in a protein. These two problems are reduced by means of FTIR difference spectroscopy, which examines the differences in absorbance before and after a physiologically relevant perturbation that alters the state of a protein (e.g., light excitation for light- sensitive proteins). After the protein is perturbed, for instance with a short laser pulse, FTIR spectra can be recorded as a function of time. The step-scan technique allows to obtain FTIR spectra from 10 ns to 100 ms, while the rapid-scan technique allow to obtain FTIR spectra from 10 ms until tens of seconds or even tens of minutes. When applied to proteins, time-resolved FTIR difference spectroscopy can provide information about changes in the protonation state of amino acid residues, chromophores/cofactors, internal water molecules and buffer molecules, as well as their time sequence. These changes in protonation state can then be used to identify proton transfer reactions and, in the case of proton- pumps, to devise a proton-pumping mechanism.

Here, first I will introduce the idea behind FTIR difference spectroscopy of proteins. Then, I will explain the time-resolved rapid-scan and step-scan techniques. After giving the foundations, I will explain how time-resolved FTIR difference spectroscopy was applied to resolve the proton transfer reactions in the light-driven proton pump bacteriorhodopsin 1–3, and the light-gated ion channel channelrhodopsin-2 4–6.

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