Louisa Reissig

A Blue-shifted Light-driven Proton Pump for Neural Silencing

Background: Light-driven proton pumps are utilized to control the neural activity. Results: We have succeeded to produce a blue-shifted proton pump. The rotation of the β-ionone ring contributes to the spectral shift. Conclusion: The designed color variant provides a tool that allows the control of neural activity by blue light. Significance: The knowledge will help to understand the color-tuning mechanism and can be utilized for optogenetics. Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λmax = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.

Three-dimensional structure and growth of myelins

After contact with water, surfactant lamellar phases (L(α)) can show spectacular interface instabilities: multibilayer tubules, so-called myelins, grow from the L(α)/water interface into the water. We have studied the shape, size, and growth of myelins in aqueous solutions of the nonionic surfactant C(12)E(3) (triethylene glycol monododecyl ether) during dissolution. We used a combination of different imaging techniques: optical microscopy providing 2-D projections of the sample and confocal microscopy offering a complete 3-D reconstruction. These techniques provide quantitative information on the shape and growth of myelins, such as their width, length, and depth profile as a function of time. The growth rate of myelins, characterized by a swelling or diffusion coefficient, was found to increase with surfactant mass fraction and, seemingly, with sample thickness. We demonstrate that myelin creaming due to buoyancy can explain the apparent dependence on sample thickness. Our experiments furthermore suggest that myelin growth is controlled by an interplay between the water mobility in the lamellar phase and the osmotic pressure difference between the lamellar phase and the contacting water.

Absorption Spectra and Photochemical Reactions in a Unique Photoactive Protein, Middle Rhodopsin MR

Photoactive proteins with cognate chromophores are widespread in organisms, and function as light-energy converters or receptors for light-signal transduction. Rhodopsins, which have retinal (vitamin A aldehyde) as their chromophore within their seven transmembrane α-helices, are classified into two groups, microbial (type-1) and animal (type-2) rhodopsins. In general, light absorption by type-1 or type-2 rhodopsins triggers a trans-cis or cis-trans isomerization of the retinal, respectively, initiating their photochemical reactions. Recently, we found a new microbial rhodopsin (middle rhodopsin, MR), binding three types of retinal isomers in its original state: all-trans, 13-cis, and 11-cis. Here, we identified the absolute absorption spectra of MR by a combination of high performance liquid chromatography (HPLC) and UV-vis spectroscopy under varying light conditions. The absorption maxima of MR with all-trans, 13-cis, or 11-cis retinal are located at 485, 479, and 495 nm, respectively. Their photocycles were analyzed by time-resolved laser spectroscopy using various laser wavelengths. In conclusion, we propose that the photocycles of MR are MR(trans) → MR(K):lifetime = 93 μs → MR(M):lifetime = 12 ms → MR, MR(13-cis) → MR(O-like):lifetime = 5.1 ms → MR, and MR(11-cis) → MR(K-like):lifetime = 8.2 μs → MR, respectively. Thus, we demonstrate that a single photoactive protein drives three independent photochemical reactions.

Influence of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I.

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.

Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin I.

Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absence of Cl(-). These phenomena have not been observed in the wild-type, or the N105Q and N105D mutants of Natronomonas pharaonis SRII (NpSRII), indicating differences in the structure and structural changes between SrSRI and NpSRII around the β-ionone ring. These differences could also be supported by the measurements of the reactivity with the water-soluble reagent azide. On the basis of these results, we discuss the structure and structural changes around the retinal chromophore in SrSRI.

A differential photodetector: Detecting light modulations using transient photocurrents

Inserting an insulating layer (I) into a conventional metal-semiconductor-metal (MSM) photodiode converts the DC photoresponse into a strong transient signal, highly applicable to modulated signal photodetection. In this study, we demonstrate the intrinsic benefits of organic MISM photodetectors, namely their effective operation under high steady-state lighting, responding only to changes in light intensity, and their ability to react to several light sources simultaneously. Furthermore, the strong interaction at the S/I interface, specific to this architecture, significantly enhances the device photoresponse, resulting in highly efficient differential photodetection, compared to a composite MSM + C devicefabricated from identical elements.

Factors Affecting the Stability and Performance of Ionic Liquid-Based Planar Transient Photodetectors

A novel planar architecture has been developed for the study of photodetectors utilizing the transient photocurrent response induced by a metal/insulator/semiconductor/metal (MISM) structured device, where the insulator is an ionic liquid (IL-MISM). Using vanadyl 2,3-naphthalocyanine, which absorbs in the communications-relevant near-infrared wavelength region (λ(max,film) ≈ 850 nm), in conjunction with C60 as a bulk heterojunction, the high capacitance of the formed electric double layers at the ionic liquid interfaces yields high charge separation efficiency within the semiconductor layer, and the minimal potential drop in the bulk ionic liquid allows the electrodes to be offset by distances of over 7 mm. Furthermore, the decrease in operational speed with increased electrode separation is beneficial for a clear modeling of the waveform of the photocurrent signal, free from the influence of measurement circuitry. Despite the use of a molecular semiconductor as the active layer in conjunction with a liquid insulating layer, devices with a stability of several days could be achieved, and the operational stability of such devices was shown to be dependent solely on the solubility of the active layer in the ionic liquid, even under atmospheric conditions. Furthermore, the greatly simplified device construction process, which does not rely on transparent electrode materials or direct electrode deposition, provides a highly reproducible platform for the study of the electronic processes within IL-MISM detectors that is largely free from architectural constraints.

SEARCH FOR SHAPE COEXISTENCE IN EVEN – EVEN STABLE MOLYBDENUM ISOTOPES USING COULOMB EXCITATION METHOD

Coulomb excitation experiments were performed with 96Mo, 98Mo and 100Mo isotopes using both beam and target excitation. As a result, quadrupole deformation parameters were deduced for two lowest-lying (ground and excited) 0+ states. Surprising shape changes of these states were observed.

Controlling the crystallinity and crystalline orientation of “shuttlecock” naphthalocyanine films for near-infrared optoelectronic applications

The thin film properties of tin(II) 2,3-naphthalocyanine (SnNPc) were interrogated and various strategies for controlling the crystallinity and crystalline orientation within the films were assessed. SnNPc is shown to crystallize in the space group P21/c (Z = 4), where the molecular arrangement consists of alternating layers of concave and convex overlap, induced by the out-of-plane Sn atoms, resulting in a 3D slipped-π-stack network structure analogous to that reported for Phase I of titanyl phthalocyanine. The thin films were studied by X-ray diffraction, atomic force microscopy and absorption spectroscopy and are highly sensitive not just to the conditions during growth, but also to substrate pre- and post-deposition treatment. While the films grown at room temperature were largely amorphous, the crystallinity was enhanced with substrate temperature, with the molecules orienting in a standing molecular geometry. A thin layer of 3,4:9,10-perlenetetracarboxylic dianhydride induces a lying molecular geometry of the same polymorph as that of the single crystal, while different polymorphs are accessible through solvent vapor annealing of amorphous films. Transient photocurrent measurements showed a dramatic improvement in photodetector device bandwidth for the lying molecular geometry, which was attributed to enhanced photoconductivity along the π-stacking axis, while solvent vapor annealing could be used to tune the photosensitivity across the near-infrared region.

Large spectral change due to amide modes of a β-sheet upon the formation of an early photointermediate of middle rhodopsin.

Rhodopsin contains retinal as the chromophore within seven transmembrane helices. Recently, we found a unique rhodopsin (middle rhodopsin, MR), which is evolutionarily located between the well-studied bacteriorhodopsin and sensory rhodopsin II, and which accommodates three retinal isomers in its ground state (the all-trans, the 13-cis, and, uniquely, the 11-cis isomers). In this study, we investigated structural changes of both the protein moiety and the retinal chromophore during photocycles of MR by time-resolved Fourier-transform infrared spectroscopy. Three photointermediates with decay time constants of 95 μs, 0.9 ms, and >~10 ms were identified by the global exponential fitting analysis. The first and third intermediates were attributed to the all-trans photocycle, in accordance with recently published results, whereas the second intermediate was likely one that was spectroscopically silent in the visible region and that was formed between the first and third states or resulted from the activation of the 13-cis isomer. By comparing light-induced difference spectra with various isotope labels in either the retinal or the protein moiety, we concluded that a β-sheet structure in the hydrophilic part was significantly altered during the all-trans photocycle of MR, which may involve an active state of the protein. This feature is characteristic of MR among microbial (type-1) rhodopsins.