It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

A technique of time-to-digital conversion is utilized in a digital demodulator for a low-power 2.4-GHz CMOS GFSK transceiver. The proposed time-to-digital converter (TDC) employs a self-sampling technique and an auto-calibration algorithm to avoid edge synchronization problems and the need of a delay-locked loop (DLL). With the TDC, a limiter and a digital demodulator can be employed simultaneously in the receiver to achieve low power consumption and high performance. Additionally, in the transmitter, the open-loop VCO modulation is adopted to save hardware and power consumption. The transmitter frequency drift in open-loop modulation and frequency offset between the receiver and the transmitter can be easily resolved by the proposed receiver architecture. All required building blocks of the proposed transceiver, except a RF matching network and a crystal, were implemented on a 4-mm 2 chip by a 0.18-m CMOS process. The receiver achieves 89-dBm sensitivity at 0.1% BER with 1-Mb/s data rate, and the transmitter delivers up to 0-dBm output power. The receiver and transmitter consume 13.3 mA and 10.7 mA, respectively, from a 1.8-V power supply. Index Terms-Complex bandpass filter, demodulator, frequency synthesizer, low-noise amplifier (LNA), open-loop VCO modulation, time-to-digital converter (TDC).

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The translocon-unassisted folding process of transmembrane domains of the microbial rhodopsins sensory rhodopsin I (HsSRI) and II (HsSRII), channelrhodopsin II (CrChR2), and bacteriorhodopsin (HsBR) during cell-free expression has been investigated by Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS). Up to now, only a limited number of rhodopsins have been expressed and folded into the functional holoprotein in cell free expression systems, while other microbial rhodopsins fail to properly bind the chromophore all-trans retinal as indicated by the missing visible absorption. SEIRAS experiments suggest that all investigated rhodopsins lead to the production of polypeptides, which are co-translationally inserted into a solid-supported lipid bilayer during the first hour after the in-vitro expression is initiated. Secondary structure analysis of the IR spectra revealed that the polypeptides form a comparable amount of α-helical structure during the initial phase of insertion into the lipid bilayer. As the process progressed (>1 h), only HsBR exhibited a further increase and association of α-helices to form a compact tertiary structure, while the helical contents of the other rhodopsins stagnated. This result suggests that the molecular reason for the unsuccessful cell-free expression of the two sensory rhodopsins and of CrChR2 is not due to the translation process, but rather to the folding process during the post-translational period. Taking our previous observation into account that HsBR fails to form a tertiary structure in the absence of its retinal, we infer that the chromophore retinal is an integral component of the compaction of the polypeptide into its tertiary structure and the formation of a fully functional protein.