Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

The Branched Photocycle of the Slow-Cycling Channelrhodopsin-2 Mutant C128T

Channelrhodopsins (ChRs) of green algae such as Chlamydomonas are used as neuroscience tools to specifically depolarize cells with light. A crude model of the ChR2 photocycle has been recently established, but details of the photoreactions are widely unknown. Here, we present the photoreactions of a slow-cycling ChR2 mutant (step function rhodopsin), with C128 replaced by threonine and 200-fold extended lifetime of the conducting-state P520. At a late state of the photocycle, a fraction of the proteins branches off into an inactive species, P380, which accumulates during prolonged illumination. At neutral pH, P380 is converted into P353, a species with a characteristic fine-structured spectrum that is interpreted as retroretinyl chromophore. The described branching reactions should be considered, when ChR is used as a neuroscience tool, especially in the case of fluorescence imaging at high light intensities.     

Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method

Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes.     

Equilibrium centrifugation of measles virus

Measles virus has been centrifuged on different density gradients. It sediments at densities of 1,20 g/cm³ in K-tartrate, of 1,18–1,21 g/cm³ in sucrose, 1,19–1,23 g/cm³ in CsCl and 1,19 g/cm³ in metrizamide gradients. Metrizamide reduced measles virus infectivity. In sucrose gradients sometimes more than one infectious peak was observed. Control Vero cells produced particles of the same densities as measles virus peaks. These peaks did contain actin as the major protein. The relevance of this finding in relation to the presence of actin in measles virus is discussed.     

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.     

A simple method for rapid purification of avian sarcoma virus supercoiled DNA by selective precipitation of infected cell chromatin.

A simple and effective method to partially purify virus-specific DNA from avian sarcoma virus-infected QT6 cells has been developed. This method consists of lysing infected cell nuclei in water followed by precipitation of the resulting chromatin with sodium chloride. More than 98% of the host cell DNA could be removed by this method without diminishing viral DNA yields. This method is equally applicable to SV40 DNA purification.