Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR.

The functional mechanism of the light-driven sodium pump rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13- retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond.

Temperature Dependence of the Krokinobacter rhodopsin 2 Kinetics.

We investigated the temperature-dependent kinetics of the light-driven Na pump Krokinobacter rhodopsin 2 (KR2) at Na-pumping conditions. The recorded microsecond flash photolysis data were subjected to detailed global target analysis, employing Eyring constraints and spectral decomposition. The analysis resulted in the kinetic rates, the composition of the different photocycle equilibria, and the spectra of the involved photointermediates.

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region.

Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time-Resolved Infrared Spectroscopy.

Ultrafast infrared transient absorption in the carbonyl vibrational region of protonated aspartate and glutamate residues in channelrhodopsin-2 from Chlamydomonas reinhardtii shows immediate protein response to retinal excitation. The observed difference bands are formed directly after the excitation on the subpicosecond time scale and were assigned to side chains in the retinal vicinity, such as D156 and E90. This finding implies an ultrafast and effective energy transfer from the retinal to its environment via hydrogen-bonded networks and reveals extraordinarily strong chromophore-protein coupling and intense interaction within the protein.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state.

From Gene to Function: Cell-Free Electrophysiological and Optical Analysis of Ion Pumps in Nanodiscs.

Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization.

Detection of argon in the coma of comet 67P/Churyumov-Gerasimenko.

Comets have been considered to be representative of icy planetesimals that may have contributed a significant fraction of the volatile inventory of the terrestrial planets. For example, comets must have brought some water to Earth. However, the magnitude of their contribution is still debated.

Glottic closing force: impact of thyroplasty on vocal cord paralysis in a pig model.

Reflex glottic closure is an essential component of a normal swallow. A lesion of the unilateral recurrent laryngeal nerve weakens this reflex response, affecting the essential protective functions of the larynx and potentially resulting in aspiration pneumonia, sepsis, or death. Thyroplasty has been advocated to reduce glottic incompetence due to unilateral vocal cord paralysis (UVCP).