Limits...
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

Different types of IR spectrometers. (A) Basic concept of a typical Fourier-transform infrared spectrometer showing light source (Globar), beam splitter, fixed and movable mirrors and single element infrared detector. Conformational changes in the sample are initiated by the trigger laser. The conversion of the sample can then be followed with a time resolution either determined by the sliding mirror movement (rapid-scan) or by the rise-time of the detector (step-scan). (B) Concept of a recently proposed dispersive device (Schade et al., 2014) with Synchrotron light source, dispersive prism and focal-plane array detector. (C) Laser based pump-probe setup. A first pulse from the pump laser starts the photoreaction. A subsequent short pulse from the probe laser probes the system. The probe pulse can be dispersed to obtain spectra; however, spectral bandwidth is determined by the duration of the probe pulse.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493399&req=5

Figure 2: Different types of IR spectrometers. (A) Basic concept of a typical Fourier-transform infrared spectrometer showing light source (Globar), beam splitter, fixed and movable mirrors and single element infrared detector. Conformational changes in the sample are initiated by the trigger laser. The conversion of the sample can then be followed with a time resolution either determined by the sliding mirror movement (rapid-scan) or by the rise-time of the detector (step-scan). (B) Concept of a recently proposed dispersive device (Schade et al., 2014) with Synchrotron light source, dispersive prism and focal-plane array detector. (C) Laser based pump-probe setup. A first pulse from the pump laser starts the photoreaction. A subsequent short pulse from the probe laser probes the system. The probe pulse can be dispersed to obtain spectra; however, spectral bandwidth is determined by the duration of the probe pulse.

Mentions: In Fourier-transform infrared (FTIR) spectrometers the light from a broadband IR-source passes an interferometer where the incident beam is split by a beam splitter. The partial beams are back-reflected to the beam splitter by two mirrors one of which is a sliding mirror introducing a position-dependent phase-shift. The beam splitter allows the partial transmission of the reflected beams to the detector, where an interference signal is recorded as a function of the optical path difference (Griffiths and De Haseth, 2007) (Figure 2A). This so-called interferogram is converted into a spectrum by a Fourier transformation (Herres and Gronholz, 1984). FTIR spectrometers benefit from high-throughput (Jacquinot), multiplex (Fellgett), and high registration precision (Connes) advantages (Perkins, 1987). The temporal resolution is only limited by the speed, sliding pathlength (corresponding to the resolution of the spectrum) and reversal-time of the movable mirror. For a spectrum of 4 cm−1 resolution, 40 ms time-resolution can be achieved (Smith and Palmer, 2002). Due to the symmetry of the interferogram around the position of equal optical path length (Δs = 0), one movement of the mirror yields two spectra by splitting the interferogram at (Δs = 0). Utilizing both forward- and backward movement of the mirror for data acquisition, a time-resolution of 10 ms is achieved. Further improvement to 5 ms (8 cm−1 resolution) was reported with the rapid-sweep method (Braiman et al., 1987). However, using sliding mechanisms means that after data acquisition the mirror has to be stopped and its direction reversed. This time-consuming process becomes significant when fast processes are investigated and the mirror is moved with high speed over a short distance. To avoid this, different types of interferometers have been utilized. For instance, a continuous rotary motion of a tilted mirror was used to measure an interferogram in less than 1 ms (4 cm−1 resolution) (Griffiths et al., 1999). However, difficulties in maintaining the alignment made an optical tilt-compensation necessary (Manning, 2002). Due to the limited time-resolution, rapid-scan FTIR is only suited to investigate the late stages of the channelrhodopsin photocycle. The conducting state can only be observed by this technique in exceptional cases, for example by cryotrapping or when slow-cycling mutants (i.e., ChR2-C128T, Berndt et al., 2009) are used (Stehfest et al., 2010).


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)

Different types of IR spectrometers. (A) Basic concept of a typical Fourier-transform infrared spectrometer showing light source (Globar), beam splitter, fixed and movable mirrors and single element infrared detector. Conformational changes in the sample are initiated by the trigger laser. The conversion of the sample can then be followed with a time resolution either determined by the sliding mirror movement (rapid-scan) or by the rise-time of the detector (step-scan). (B) Concept of a recently proposed dispersive device (Schade et al., 2014) with Synchrotron light source, dispersive prism and focal-plane array detector. (C) Laser based pump-probe setup. A first pulse from the pump laser starts the photoreaction. A subsequent short pulse from the probe laser probes the system. The probe pulse can be dispersed to obtain spectra; however, spectral bandwidth is determined by the duration of the probe pulse.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493399&req=5

Figure 2: Different types of IR spectrometers. (A) Basic concept of a typical Fourier-transform infrared spectrometer showing light source (Globar), beam splitter, fixed and movable mirrors and single element infrared detector. Conformational changes in the sample are initiated by the trigger laser. The conversion of the sample can then be followed with a time resolution either determined by the sliding mirror movement (rapid-scan) or by the rise-time of the detector (step-scan). (B) Concept of a recently proposed dispersive device (Schade et al., 2014) with Synchrotron light source, dispersive prism and focal-plane array detector. (C) Laser based pump-probe setup. A first pulse from the pump laser starts the photoreaction. A subsequent short pulse from the probe laser probes the system. The probe pulse can be dispersed to obtain spectra; however, spectral bandwidth is determined by the duration of the probe pulse.
Mentions: In Fourier-transform infrared (FTIR) spectrometers the light from a broadband IR-source passes an interferometer where the incident beam is split by a beam splitter. The partial beams are back-reflected to the beam splitter by two mirrors one of which is a sliding mirror introducing a position-dependent phase-shift. The beam splitter allows the partial transmission of the reflected beams to the detector, where an interference signal is recorded as a function of the optical path difference (Griffiths and De Haseth, 2007) (Figure 2A). This so-called interferogram is converted into a spectrum by a Fourier transformation (Herres and Gronholz, 1984). FTIR spectrometers benefit from high-throughput (Jacquinot), multiplex (Fellgett), and high registration precision (Connes) advantages (Perkins, 1987). The temporal resolution is only limited by the speed, sliding pathlength (corresponding to the resolution of the spectrum) and reversal-time of the movable mirror. For a spectrum of 4 cm−1 resolution, 40 ms time-resolution can be achieved (Smith and Palmer, 2002). Due to the symmetry of the interferogram around the position of equal optical path length (Δs = 0), one movement of the mirror yields two spectra by splitting the interferogram at (Δs = 0). Utilizing both forward- and backward movement of the mirror for data acquisition, a time-resolution of 10 ms is achieved. Further improvement to 5 ms (8 cm−1 resolution) was reported with the rapid-sweep method (Braiman et al., 1987). However, using sliding mechanisms means that after data acquisition the mirror has to be stopped and its direction reversed. This time-consuming process becomes significant when fast processes are investigated and the mirror is moved with high speed over a short distance. To avoid this, different types of interferometers have been utilized. For instance, a continuous rotary motion of a tilted mirror was used to measure an interferogram in less than 1 ms (4 cm−1 resolution) (Griffiths et al., 1999). However, difficulties in maintaining the alignment made an optical tilt-compensation necessary (Manning, 2002). Due to the limited time-resolution, rapid-scan FTIR is only suited to investigate the late stages of the channelrhodopsin photocycle. The conducting state can only be observed by this technique in exceptional cases, for example by cryotrapping or when slow-cycling mutants (i.e., ChR2-C128T, Berndt et al., 2009) are used (Stehfest et al., 2010).

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