Disc antenna enhanced infrared spectroscopy: From felf-assembled monolayers to membrane proteins

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 104 giving rise to a detection limit <1 femtomol (10–15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region

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

Photoexcitation of the P4480 state induces a secondary photocycle that potentially desensitizes channelrhodopsin-2

Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P3520 intermediate of the primary all-trans photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P4480, the last intermediate of the all-trans photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I3530. Our results indicate that the D2 and O2 states correspond to the P4480 and I3530 intermediates, connecting desensitization of ChR2 with the photochemical properties of the P4480 intermediate

wavelength dependent photoreactions induced by ground-state heterogeneity

The primary photodynamics of channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) was investigated by VIS-pump supercontinuum probe experiments from femtoseconds to 100 picoseconds. In contrast to reported experiments on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), we found a clear dependence of the photoreaction dynamics on varying the excitation wavelength. Upon excitation at 500 and at 550 nm we detected different bleaching bands, and spectrally distinct photoproduct absorptions in the first picoseconds. We assign the former to the ground-state heterogeneity of a mixture of 13-cis and all-trans retinal maximally absorbing around 480 and 540 nm, respectively. At 550 nm, all-trans retinal of the ground state is almost exclusively excited. Here, we found a fast all-trans to 13-cis isomerization process to a hot and spectrally broad P1 photoproduct with a time constant of (100 ± 50) fs, followed by photoproduct relaxation with time constants of (500 ± 100) fs and (5 ± 1) ps. The remaining fraction relaxes back to the parent ground state with time constants of (500 ± 100) fs and (5 ± 1) ps. Upon excitation at 500 nm a mixture of both chromophore conformations is excited, resulting in overlapping reaction dynamics with additional time constants of <300 fs, (1.8 ± 0.3) ps and (90 ± 25) ps. A new photoproduct Q is formed absorbing at around 600 nm. Strong coherent oscillatory signals were found pertaining up to several picoseconds. We determined low frequency modes around 200 cm−1, similar to those reported for bacteriorhodopsin

Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump - IR-Probe Spectroscopy and Computational Methods

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivit

Time-resolved photoacoustics of channelrhodopsins: early energetics and light-driven volume changes

In biological photoreceptors, the energy stored in early transient species is a key feature to drive the photocycle or a chain of reactions. Time-resolved photoacoustics (PA) can explore the energy landscape of transient species formed within few ns after photoexcitation, as well as volumetric changes (Delta V) of these intermediates with respect to the parental state. In this work, PA identified these important parameters for several channelrhodopsins, namely CaChR1 from Chlamydomonas augustae and CrChR2 from Chlamydomonas reinhardtii and various variants. PA has access to the sub-ns formation of the early photoproduct P1 and to its relaxation, provided that this latter process occurs within a few mu s. We found that Delta V-P1 for CaChR1 is ca. 12 mL/mol, while it is much smaller for CrChR2 (4.7 mL/mol) and for H. salinarum bacteriorhodopsin (HsBR, Delta V-K = 2.8 mL/mol). PA experiments on variants strongly indicate that part of this large Delta V-P1 value for CaChR1 is caused by the protonation dynamics of the Schiff base counterion complex involving E169 and D299. PA data further show that the energy level of P1 is higher in CrChR2 (ca. 96 kJ/mol) than in CaChr1 (ca. 46 kJ/mol), comparable to the energy level of the K state of HsBR (60 kJ/mol). Instrumental to gain these molecular values from the raw PA data was the estimation of the quantum yield (Phi) for P1 formation via transient spectroscopy; for both channelrhodopsins, Phi(P2) was evaluated as ca. 0.4

Multimedia in learning and instruction. State of the art and research perspectives

Mit den neuen Medien sind Hoffnungen auf eine Verbesserung der Lernwirksamkeit von Unterricht verbunden. Der Beitrag gibt einen zusammenfassenden Überblick über den Stand der empirischen Forschung zum Lernen mit neuen Medien. Berücksichtigung finden Erkenntnisse kognitionspsychologischer, instruktionspsychologischer und mediendidaktischer Provenienz. Im Anschluss an eine methodenkritische Einschätzung der Forschungslage erfolgen zunächst Aussagen über die Lernwirksamkeit neuer Medien im Hinblick auf ihre Gestaltungsmerkmale (i. e. das Zusammenspiel von Text, Bild und Ton, das Zusammenspiel der Codierung von Lernaufgaben und Testaufgaben sowie Ablaufgeschwindigkeit und Strukturierung der präsentierten Informationen). Anschließend wird auf die Lernwirksamkeit der neuen Medien unter Beachtung von Lehr-Lernzielen und von Persönlichkeitsmerkmalen der Schüler (i. e. Interessen und Einstellungen, themenspezifische Vorkenntnisse und medienspezifische Fertigkeiten sowie Lernstrategien) sowie im Hinblick auf mögliche instruktionale Unterstützung näher eingegangen. Auf dieser Basis werden Forschungsperspektiven entwickelt, die insbesondere auf eine Ergänzung der dominierenden Laborstudien um Forschung im realen Alltag von Lehrerinnen und Lehrer zielen. (DIPF/Orig.

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far

pH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm−1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin