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Time-resolved infrared spectroscopic techniques as applied to channelrhodopsin.

Ritter E, Puskar L, Bartl FJ, Aziz EF, Hegemann P, Schade U - Front Mol Biosci (2015)

Bottom Line: To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary.This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins.We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.

View Article: PubMed Central - PubMed

Affiliation: Experimentelle Biophysik, Institut für Biologie, Humboldt-Universität zu Berlin Berlin, Germany.

ABSTRACT
Among optogenetic tools, channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.

No MeSH data available.


Related in: MedlinePlus

Infrared spectroscopy of Channelrhodopsin. The absorption spectrum (gray) of retinal proteins like Channelrhodopsin-2 reconstituted in lipid vesicles shows bands associated with the lipid environment and protonated carboxyl groups (~1700–1800 cm−1), water (1644 cm−1) and the overall helical structure of the protein (amide I ~1650 cm−1; amide II ~1550 cm−1). Note, that the lipid vesicles allow a very dense packing of the protein in the cuvette thus reducing the water content. Light induced alterations are represented by the difference spectrum (black), where negative bands (blue) occur due to the dark state while positive bands (red) are due to the illuminated state, achieved by illumination with blue (480 nm) light. The spectrum was recorded at cryogenic conditions where a mixture of species, including the Schiff base deprotonated state and the conducting state is observed. Note that, while total absorbance is in the order of 0.9 OD (left scale, gray), largest changes in the difference spectrum are within 0.004 OD (right scale, black). In the picture, some bands assigned so far to their structural counterparts are marked. For details of the band assignments, see (Eisenhauer et al., 2012; Lórenz-Fonfría et al., 2013, 2015; Kuhne et al., 2015).
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Figure 1: Infrared spectroscopy of Channelrhodopsin. The absorption spectrum (gray) of retinal proteins like Channelrhodopsin-2 reconstituted in lipid vesicles shows bands associated with the lipid environment and protonated carboxyl groups (~1700–1800 cm−1), water (1644 cm−1) and the overall helical structure of the protein (amide I ~1650 cm−1; amide II ~1550 cm−1). Note, that the lipid vesicles allow a very dense packing of the protein in the cuvette thus reducing the water content. Light induced alterations are represented by the difference spectrum (black), where negative bands (blue) occur due to the dark state while positive bands (red) are due to the illuminated state, achieved by illumination with blue (480 nm) light. The spectrum was recorded at cryogenic conditions where a mixture of species, including the Schiff base deprotonated state and the conducting state is observed. Note that, while total absorbance is in the order of 0.9 OD (left scale, gray), largest changes in the difference spectrum are within 0.004 OD (right scale, black). In the picture, some bands assigned so far to their structural counterparts are marked. For details of the band assignments, see (Eisenhauer et al., 2012; Lórenz-Fonfría et al., 2013, 2015; Kuhne et al., 2015).

Mentions: IR-spectroscopy was among the first techniques used to obtain structural information on the channelrhodopsin photocycle (Ritter et al., 2008; Radu et al., 2009). Since 2008, several bands have been assigned by biophysical methods such as site-directed mutagenesis, H2O/2H2O exchange or isotopic labeling. Figure 1 (light gray) shows the IR-spectrum of Channelrhodopsin-2 with some important bands marked. For example, its overall helical structure is typically discerned from the amide I and II bands (~1660 and ~1550 cm−1) (Bandekar and Krimm, 1979; Byler and Susi, 1986; Goormaghtigh, 1990). However, when only the modes that undergo a change during conformational alterations of the protein are of interest, the difference spectrum calculated by subtracting the spectrum of the functional (illuminated) state from the spectrum of the resting (dark) state is used (Figure 1, black line). Hereby structural changes connected with the pre-formation, opening or closing of the pore become visible. The band at 1661 cm−1 indicates conformational changes of the protein, the band pattern between 1100 and 1300 cm−1 reflects the all-trans/13-cis chromophore isomerization (Nack et al., 2009; Bruun et al., 2011), whereas changes in hydrogen bonding and proton transfers of functional aspartates and glutamates are seen between 1700 and 1800 cm−1. The protonation states and hydrogen bonding of the Schiff base counter ions E123 and D253 (1760 cm−1) and the proton donor D156 (1737 and 1760 cm−1) can be directly observed as well as the protonation state of E90 which, as a part of the central gate, plays a role in channel selectivity. For further band assignments see for instance (Kuhne et al., 2015; Lórenz-Fonfría et al., 2015) and citations therein.


Time-resolved infrared spectroscopic techniques as applied to channelrhodopsin.

Ritter E, Puskar L, Bartl FJ, Aziz EF, Hegemann P, Schade U - Front Mol Biosci (2015)

Infrared spectroscopy of Channelrhodopsin. The absorption spectrum (gray) of retinal proteins like Channelrhodopsin-2 reconstituted in lipid vesicles shows bands associated with the lipid environment and protonated carboxyl groups (~1700–1800 cm−1), water (1644 cm−1) and the overall helical structure of the protein (amide I ~1650 cm−1; amide II ~1550 cm−1). Note, that the lipid vesicles allow a very dense packing of the protein in the cuvette thus reducing the water content. Light induced alterations are represented by the difference spectrum (black), where negative bands (blue) occur due to the dark state while positive bands (red) are due to the illuminated state, achieved by illumination with blue (480 nm) light. The spectrum was recorded at cryogenic conditions where a mixture of species, including the Schiff base deprotonated state and the conducting state is observed. Note that, while total absorbance is in the order of 0.9 OD (left scale, gray), largest changes in the difference spectrum are within 0.004 OD (right scale, black). In the picture, some bands assigned so far to their structural counterparts are marked. For details of the band assignments, see (Eisenhauer et al., 2012; Lórenz-Fonfría et al., 2013, 2015; Kuhne et al., 2015).
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Infrared spectroscopy of Channelrhodopsin. The absorption spectrum (gray) of retinal proteins like Channelrhodopsin-2 reconstituted in lipid vesicles shows bands associated with the lipid environment and protonated carboxyl groups (~1700–1800 cm−1), water (1644 cm−1) and the overall helical structure of the protein (amide I ~1650 cm−1; amide II ~1550 cm−1). Note, that the lipid vesicles allow a very dense packing of the protein in the cuvette thus reducing the water content. Light induced alterations are represented by the difference spectrum (black), where negative bands (blue) occur due to the dark state while positive bands (red) are due to the illuminated state, achieved by illumination with blue (480 nm) light. The spectrum was recorded at cryogenic conditions where a mixture of species, including the Schiff base deprotonated state and the conducting state is observed. Note that, while total absorbance is in the order of 0.9 OD (left scale, gray), largest changes in the difference spectrum are within 0.004 OD (right scale, black). In the picture, some bands assigned so far to their structural counterparts are marked. For details of the band assignments, see (Eisenhauer et al., 2012; Lórenz-Fonfría et al., 2013, 2015; Kuhne et al., 2015).
Mentions: IR-spectroscopy was among the first techniques used to obtain structural information on the channelrhodopsin photocycle (Ritter et al., 2008; Radu et al., 2009). Since 2008, several bands have been assigned by biophysical methods such as site-directed mutagenesis, H2O/2H2O exchange or isotopic labeling. Figure 1 (light gray) shows the IR-spectrum of Channelrhodopsin-2 with some important bands marked. For example, its overall helical structure is typically discerned from the amide I and II bands (~1660 and ~1550 cm−1) (Bandekar and Krimm, 1979; Byler and Susi, 1986; Goormaghtigh, 1990). However, when only the modes that undergo a change during conformational alterations of the protein are of interest, the difference spectrum calculated by subtracting the spectrum of the functional (illuminated) state from the spectrum of the resting (dark) state is used (Figure 1, black line). Hereby structural changes connected with the pre-formation, opening or closing of the pore become visible. The band at 1661 cm−1 indicates conformational changes of the protein, the band pattern between 1100 and 1300 cm−1 reflects the all-trans/13-cis chromophore isomerization (Nack et al., 2009; Bruun et al., 2011), whereas changes in hydrogen bonding and proton transfers of functional aspartates and glutamates are seen between 1700 and 1800 cm−1. The protonation states and hydrogen bonding of the Schiff base counter ions E123 and D253 (1760 cm−1) and the proton donor D156 (1737 and 1760 cm−1) can be directly observed as well as the protonation state of E90 which, as a part of the central gate, plays a role in channel selectivity. For further band assignments see for instance (Kuhne et al., 2015; Lórenz-Fonfría et al., 2015) and citations therein.

Bottom Line: To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary.This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins.We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.

View Article: PubMed Central - PubMed

Affiliation: Experimentelle Biophysik, Institut für Biologie, Humboldt-Universität zu Berlin Berlin, Germany.

ABSTRACT
Among optogenetic tools, channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.

No MeSH data available.


Related in: MedlinePlus