The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane

AbstractWe measured the flash-induced absorption anisotropies of mutant bacteriorhodopsin (bR), D96N, in the purple membrane suspension. The measured anisotropy decay at 410 nm differed from that at 570 nm. These wavelength-dependent anisotropies show that the motion of absorption dipole of non-excited bR is faster than that of M-intermediate. The motion of non-excited bR is considered as the rotational motion of whole protein in the purple membrane. This fact suggests that the photo-excitation induces the conformational change of the protein and/or the inter-protein interaction within the membrane, which prevents the motion of M-intermediate

Photo-induced Proton Transfers of Microbial Rhodopsins

Molecular Photochemistry - Various Aspect

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pK(a)=3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins

Photochemical characterization of actinorhodopsin and its functional existence in the natural host

Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed lambda(max) at 534 nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P-620, could be assigned to the 0 intermediate in other H+ pumps. In contrast to conventional 0, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH 7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P-650, while the other first releases H+ prior to H+ uptake during P-620 formation. (C) 2016 Elsevier B.V. All rights reserved

Direct Detection of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin

For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle

All-optical switching in Pharaonis phoborhodopsin protein molecules.

Low-power all-optical switching with pharaonis phoborhodopsin (ppR) protein is demonstrated based on nonlinear excited-state absorption at different wavelengths. A modulating pulsed 532-nm laser beam is shown to switch the transmission of a continuous-wave signal light beam at: 1) 390 nm; 2) 500 nm; 3) 560 nm; and 4) 600 nm, respectively. Simulations based on the rate equation approach considering all seven states in the ppR photocycle are in good agreement with experimental results. It is shown that the switching characteristics at 560 and 600 nm, respectively, can exhibit negative to positive switching. The switching characteristics at 500 nm can be inverted by increasing the signal beam intensity. The profile of switched signal beam is also sensitive to the modulating pulse frequency and signal beam intensity and wavelength. The switching characteristics are also shown to be sensitive to the lifetimes of$mmb pbf pR_bf M$and$mmb pbf pR_bf O$intermediates. The results show the applicability of ppR as a low-power wavelength tunable all-optical switch

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump

Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H+ and Na+ pumps and an inward Cl- pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H+ pump bacteriorhodopsin, which was converted to a Cl- pump in 1995 by a single amino acid replacement. In this study we report the first success of the reverse conversion from a Cl- pump to a H+ pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl- pump and belongs to a cluster that is far distant from the known Cl- pumps. With a single amino acid replacement, MrHR is converted to a H+ pump in which dissociable residues function almost completely in the H+ relay reactions. MrHR most likely evolved from a H+ pump, but it has not yet been highly optimized into a mature Cl- pump

Salt bridge in the conserved His-Asp cluster in Gloeobacter rhodopsin contributes to trimer formation

Gloeobacter rhodopsin (GR) is a eubacterial proton pump having a highly conserved histidine near the retinal Schiff base counter-ion, aspartate. Various interactions between His and Asp of the eubacterial proton pump have been reported. Here, we showed the pH-dependent trimer/monomer transition of GR in the presence of dodecyl-β-D-maltoside by size-exclusion chromatography. The pH dependence was closely related to the protonation state of the counter-ion, Asp121. For the H87M mutant, pH dependence disappeared and a monomer became dominant. We concluded that the formation or breaking of the salt bridge between His87 and Asp121 inside the protein changes the quaternary structure