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Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.

ABSTRACT: Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.

SUBMITTER: Ritter E

PROVIDER: S-EPMC3259888 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.

Ritter Eglof E Stehfest Katja K Berndt Andre A Hegemann Peter P Bartl Franz J FJ

The Journal of biological chemistry 20081016 50

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on t ...[more]

PMID: 18927082

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Project description:Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy.

Dataset Information

Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.

Publications

Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.

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