Bimodal activation of different neuron classes with the spectrally red-shifted channelrhodopsin chimera C1V1 in Caenorhabditis elegans

The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes—T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V—have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products

Ion Selectivity and Competition in Channelrhodopsins

AbstractChannelrhodopsins are light-gated ion channels of green algae. They are widely used for the analysis of neuronal networks using light in the emerging field of optogenetics. Under steady-state light conditions, the two open states, O1 and O2, mediate the photocurrents with different ion conductance and selectivity. To understand the conducting process as well as its optogenetic applications, it is important to study ion binding and transport of this promiscuous cation channel. Here, we present an enzyme kinetic algorithm that allowed us to calculate the ion composition of the initial and steady-state photocurrents for multication media. The approach is based on current-voltage relations determined for the individual ions H+, Na+, Ca2+, and Mg2+. We identify and quantify the widely different competition of the ions in wild-type channelrhodopsin-2 and two high-performing channelrhodopsin variants CatCh+ and C1V1. Both variants show enhanced Ca2+ conductance, but only CatCh+ displays high steady-state Ca2+ currents at neutral pH due to reduced H+ competition and low inactivation. We demonstrate that for optogenetic applications, one should always take into account that the variable equilibria of the two open states depend on light intensity, voltage, and the ionic composition of the medium

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at lambda(max) approximate to 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580(I) and Ret_580(II). Photoexcitation of Ret_580(I) resulted in barrier-involved isomerization to Ret_540 (quantum yield approximate to 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580(I) and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime approximate to 2 h). Photoexcitation of Ret_580(II) resulted in barrier-involved isomerization to Ret_640 (quantum yield approximate to 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580(II) and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime approximate to 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580(I) and Ret_580(II) were developed and photocyle parameters were determined

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site

Tailor-Made Silver Release Properties of Silver-Containing Functional Plasma Polymer Coatings Adjusted Through a Macroscopic Kinetics Approach

Combining a functional plasma polymer matrix with antibacterially active silver (Ag) within a nanocomposite structure allows secure production and applications in various fields, especially in the medical sector. Therefore, nitrogen or oxygen containing hydrocarbon plasma polymers and Ag nanoparticles were simultaneously deposited. Functional groups such as amino or carboxylic groups as well as an adjusted amount of Ag can be incorporated into the growing films by controlling the plasma deposition properties. For this purpose, macroscopic kinetics were used to characterise the deposition behaviour also as a base for possible industrial up-scaling. XPS and ICP-OES were used to analyse the chemical composition of the polymer-Ag nanocomposites and the Ag content which could be incorporated depending on the plasma process conditions. Finally, the Ag release was determined in bi-distilled water for classification and comparison with the antibacterial properties. The antibacterial effect of the polymer-Ag nanocomposites was proofed with the gram− strain Pseudomonas aeruginosa PAO1 and the gram+ strain Staphylococcus aureus (ST12 Group) showing a clear efficacy dependence on the amount of released Ag and the possibility for tailor-made antibacterial active plasma film

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 +/- 1 degrees C) and at refrigerator temperature (3 +/- 1 degrees C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 +/- 3 degrees C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 degrees C, 22 days at 3 degrees C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S-0 ground-state isomerization

Algen, Froscheier und Putzfimmel bei Fliegen

Zu den wichtigsten Signalmolekülen in lebenden Zellen gehört cAMP, das grundlegende Prozesse steuert. Wir haben kürzlich mithilfe eines Algenproteins eine neue Methode entwickelt, cAMP durch kurze Lichtblitze in tierischen Zellen und lebenden Tieren zu erzeugen. Bei Fliegen lassen sich damit Verhaltensänderungen an- und wieder abschalten. Die besonders hohe zeitliche Auflösung dieses neuen Lichtgesteuerten „Werkzeugs“ sollte es ermöglichen, die Rolle von cAMP bei komplexen biologischen Fragestellungen besser zu verstehen.Not Reviewe