structure and mechanism of a light-gated cation channel

The new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10− 12 s to 10− 3 s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed

Channelrhodopsin unchained: Structure and mechanism of a light-gated cation channel

AbstractThe new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10−12s to 10−3s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2 380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of “dangling” water molecules were detected in two different states of the protein using H2 18O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm−1) and with the other O-H group medium (3440 cm−1) to moderately strongly (3300 cm−1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the “dangling” water molecules within the protein, providing a better understanding of their functional relevance in CaChR1

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time- resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm−1. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2 380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of ¿dangling¿ water molecules were detected in two different states of the protein using H2 18O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm−1) and with the other O-H group medium (3440 cm−1) to moderately strongly (3300 cm−1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the ¿dangling¿ water molecules within the protein, providing a better understanding of their functional relevance in CaChR1

Análisis de la estructura del transportador ADP/ATP por espectroscopia de infrarrojo utilizando métodos matemáticos de estrechamiento y ajuste de bandas

Consultable des del TDXTítol obtingut de la portada digitalitzad

The Grateful Infrared: Sequential Protein Structural Changes Resolved by Infrared Difference Spectroscopy

Kottke T, Lórenz-Fonfría VA, Heberle J. The Grateful Infrared: Sequential Protein Structural Changes Resolved by Infrared Difference Spectroscopy. The Journal of Physical Chemistry B. 2017;121(2):335-350

pH titration monitored by quantum cascade laser-based vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra of aqueous solutions of proline were recorded in the course of titrations from basic to acidic pH using a spectrometer equipped with a quantum cascade laser (QCL) as an infrared light source in the spectral range from 1320 to 1220 cm1. The pH-dependent spectra were analyzed by singular value decomposition and global fitting of a two-pK Henderson-Hasselbalch model. The analysis delivered relative fractions of the three different protonation species. Their agreement with the relative fractions obtained from performing the same analysis on pH-dependent Fourier transform infrared (FT-IR) and QCL-IR spectra validates the quantitative results from QCL-VCD. Global fitting of the pH-dependent VCD spectra of l-proline allowed for extraction of pure spectra corresponding to anionic, zwitterionic, and cationic l-proline. From a static experiment, only pure spectra of the zwitterion would be accessible in a straightforward way. A comparison to VCD spectra calculated for all three species led to assignment of vibrational modes that are characteristic for the respective protonation states. The study demonstrates the applicability of QCL-VCD both for quantitative evaluation and for qualitative interpretation of dynamic processes in aqueous solutions