Ionela Radu

Conformational Changes of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (P(1) state) and succeeding (P(4)) the conductive state of the channel (P(3)) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D156, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime of P(3) is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P(3) state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channels conductance.

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.

Ultrafast infrared spectroscopy on channelrhodopsin-2 reveals efficient energy transfer from the retinal chromophore to the protein.

The primary reaction dynamics of channelrhodopsin-2 was investigated using femtosecond vis-pump/mid-IR probe spectroscopy. Due to the fast deactivation of the excited state in channelrhodopsin-2, it is possible to observe the direct impact of retinal isomerization on the protein surrounding. We show that the dominant negative band at 1665 cm(-1) tentatively assigned to an amide I vibration is developed with a time constant of 0.5 ps. Also a variety of side-chain vibrations are formed or intensified on this time scale. The comparison of the light-induced FT-IR spectra of channelrhodopsin-2 in H2O and D2O at 80 K enabled us to tentatively identify the contribution of Arg side chain(s). The subsequently observed decay of nearly the whole difference pattern has a particularly high impact on the C═C and C═N stretching vibrations of the retinal. This suggests that the underlying mechanism describes a cooling process in which the excess energy is redirected toward the retinal surrounding, e.g., the protein and functional water molecules. The pronounced protein contributions in comparison to other rhodopsins point to a very efficient energy redistribution in channelrhodopsin-2.

Kinetics of Proton Release and Uptake by Channelrhodopsin-2

Electrophysiological experiments showed that the light‐activated cation channel channelrhodopsin‐2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water‐soluble pH‐indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P 3 520 intermediate. As the P 3 520 state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.

Time-resolved FT-IR Spectroscopy of Membrane Proteins

Time-resolved Fourier transform infrared spectroscopy (FT-IR) offers distinct advantages concerning restrictions pertinent to biomolecules. In particular, it is possible to monitor the temporal evolution of the reaction mechanism of complex machineries as membrane proteins, where other techniques encounter significant experimental difficulties. Here, we present the classical principles and experimental realizations of time-resolved FT-IR spectroscopy together with recent developments employed in our laboratory. Examples from applications to retinal proteins are reviewed that underline the impact of time-resolved FT-IR spectroscopy on the understanding of protein reactions on the level of single bonds.

Signal relay from sensory rhodopsin I to the cognate transducer HtrI: assessing the critical change in hydrogen-bonding between Tyr-210 and Asn-53.

Sensory rhodopsin I (SRI) from Halobacterium salinarum mediates both positive and negative phototaxis in a light-dependent manner. SRI photoactivation elicits extensive structural changes which are transmitted to the cognate transducer (HtrI). The atomic structure of the SRI-HtrI complex has not been solved yet and, therefore, details on the interaction which define the binding site between receptor and transducer are missing. The related complex SRII-HtrII from Natronobacterium pharaonis exhibits a hydrogen bond between the receptor Y199 and transducer N54. This bond has been suggested to mediate signal relay in the SRII-HtrII system. Our previous results on the SRI-HtrI complex indicated that HtrI N53 forms a hydrogen bond at the cytoplasm-proximity of the membrane. Here, based on kinetic and spectroscopic data, we demonstrate that Y210 of SRI is functionally significant for the signal relay in the SRI-HtrI complex. Each of the tyrosine residues Y197, Y208, Y210 and Y213 were conservatively exchanged for phenylalanine but only the Y210F mutation led to the disappearance of the infrared band of the terminal amide C=O of N53. From this FT-IR spectroscopic result, we conclude that Y210 of SRI and N53 of HtrI interact via a hydrogen bond which is crucial for the signal transfer from the light receptor to the transducer.

Time-Resolved FT-IR Spectroscopy for the Elucidation of Protein Function

Time-resolved Fourier transform infrared spectroscopy (FT-IR) has been proven to be an excellent method with important applications in bioscience. In particular, it is possible to monitor the temporal evolution of the reaction mechanism of complex machineries as membrane proteins, where other techniques encounter significant experimental difficulties. Here, we summarize the classical principles and experimental realizations of time-resolved FT-IR spectroscopy together with new developments realized in our laboratory. Examples from applications to retinal proteins are reviewed that showcase the impact of time-resolved FT-IR spectroscopy on the understanding of protein reactions on the level of single bonds.