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Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations. [ABSTRACT FROM AUTHOR]

Infrared difference spectroscopy measurements have shown that the -OH group of Thr118 from bovine rhodopsin (Rho) has displayed a slow but measurable hydrogen/deuterium exchange (HDX) under native conditions. A quantitative two-step model in which the dark activation of Rho is triggered by thermal isomerization of the retinal in a transiently opened RBP and the absolute sensitivity threshold of the visual system is limited by structural fluctuations of the chromophore binding pocket rather than in the chromophore itself.

Attenuated total reflectance infrared (ATR-IR) difference spectra with polarized light is obtained for showing the simplification of the study and interpretation of the structural changes in membrane proteins upon substrate binding. The combination of a maximum-entropy band-narrowing method and spectroscopic rules has helped in identifying changes in the tilt of some helices in the secondary transporter melibiose permease following melibiose binding in the presence of sodium.

The transition between two M substates is characterized by time-resolved Fourier transform infrared (FT-IR) spectroscopy under functional conditions by using a novel approach for analyzing kinetic data. Vectorial proton transport is guaranteed by the coupled proton affinity changes resulting from the proton release group (PRG) deprotonation, which has favored an affinity-based over an accessibility-based switch mechanism.

The first high-quality room-temperature time-resolved infrared (FTIR) spectrum of the bacteriorhodopsin (bR) photocycle is reported. The study presents the differences in internal water molecules between room- and low-temperature spectra for the L and M intermediates with respect to the bR ground state.

Fourier transform infrared (FTIR) difference spectroscopy is used to study the substrate-induced structural changes of the melibiose permease (Me1B) from Escherichia coli reconstituted into liposomes. The analysis shows that the changes in the protonation state and/or in the environment of given carboxylic residues are induced by melibiose-Me1B interaction in the presence of cations.

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 μs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm-1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin. [ABSTRACT FROM AUTHOR]