Exploiting Photoisomerization: Spectroscopy on a Carotenoid Sensor and Retinal Proteins

Light-based methodologies enjoy popularity due to their non-invasive nature. In particular in the field of optogenetics, where genetic targeting of neurons permits not only simultaneous imaging of a large number of cells but also optical control of neuronal activity. For this, ion channels or pumps are inserted into the membrane which are activated by light. A deep biophysical understanding of the optogenetic systems is key for their successful application. In this thesis, I present a new member in the family of organic voltage sensors. I demonstrate that in a single lipid bilayer environment, the carotenoid Zeaxanthin has a linear and reversible spectral Raman response to an electric field applied across the membrane. The underlying mechanism is an increased photoisomerization rate resulting in a higher 13-cis population which is detected via a characteristic vibrational band at 1130 cm−1. Channelrhodopsin-2 (ChR2) is a frequently used protein in optogenetics to silence neuronal activity. By variation of amino acid side chains, we found experimental evidence for ground-state heterogeneity in the hydrogen bond interactions of the retinal protonated Schiff base (PSB). We have identified with Raman spectroscopy two spectral components of the C=N–H mode of the PSB at 1661 and 1665 cm−1, representing hydrogen bonds to different amino acid side chains. These two interactions of the PSB could be essential for a voltage-sensing mechanism in ChR2. In a pioneering approach we combined time-resolved absorption spectroscopy with serial femtosecond X-ray crystallography to scrutinize mechanistic details of sodium pumping in Krokinobacter eikastus rhodopsin 2 (KR2). Using an infrared-emitting quantum cascade laser (QCL), we verified that crystalline KR2 exhibits reaction kinetics similar to those observed in its detergent solubilized form. Hereupon, we have identified a previously proposed transient sodium binding site during the O intermediate where the sodium is coordinated by the amino acid side chains of N112 and D251. The findings regarding the ion transport mechanism in KR2 will facilitate the design of protein variants for an optogenetic application. Bistable G-protein coupled receptors (GPCRs) have two thermally stable conformations and are a promising class of rhodopsins which have the potential to serve as an optogenetic switch. We were able to conduct a first biophysical characterization of the invertebrate jumping spider rhodopsin-1 (JSR1). We propose a model of the two-photon reaction based on spectroscopic results. During these reactions, the Schiff base stays protonated implying that a deprotonation is not a prerequisite for the function of bistable GPCRs. A proposed mediating water molecule as part of the counterion complex in the inactive conformation is identified by Raman spectroscopy and later confirmed by an X-ray crystallographic structure. In conclusion, this thesis provides insights into the mechanistic details of established and upcoming optogenetic tools. These results will help to adapt their biophysical properties better suiting the needs of application.Lichtbasierende Methoden erfreuen sich aufgrund ihrer nicht-invasiven Eigenschaft großer Beliebtheit. Im Besonderen in der Optogenetik, wo Neuronen genetisch modifiziert werden um nicht nur die simultane Beobachtung einer großen Anzahl von Neuronen, sondern auch optische Kontrolle von neuronaler Aktivität zu ermöglichen. Hierzu werden Ionenkanäle oder -pumpen in die Membran gebracht, die durch Licht aktiviert werden können. Ein tiefes Verständnis von optogenetischen Systemen ist eine Schlüsselvoraussetzung für eine erfolgreiche Anwendung. In dieser Arbeit präsentiere ich einen Neuzugang in die Familie der organischen Spannungssensoren. Ich demonstriere, dass das Karotenoid Zeaxanthin, eingebracht in eine einzelne Lipiddoppelschicht, eine lineare und reversible Reaktion zeigt, wenn ein elektrisches Feld über die Membran angelegt wird. Der zugrunde liegende Mechanismus ist eine größere Population an 13-cis Isomeren, hervorgerufen durch eine erhöhte Photoisomerationsrate. Dies führt zu einem Anwachsen einer charakteristischen Vibrationsbande bei 1130 cm−1. Kanalrhodopsin-2 (ChR2) wird regelmäßig in der Optogentik genutzt um neuronale Aktivität zu verhindern. Durch Variation von Aminosäurenseitenketten liefern wir Beweise für eine Heterogenität in der Wasserstoffbrückeninteraktion der protonierten Schiffschen Base (PSB) im Grundzustand. Wir konnten mit Raman Spektroskopie zwei spektrale Komponenten in der PSB Vibrationsmode (C=N–H) bei 1661 und 1665 cm−1 identifizieren, die jeweils eine Wasserstoffbrücke zu einer anderen Aminosäurenseitenkette darstellen. Diese zwei Interaktionen könnten von Bedeutung für einen Spannungsmessungsmechanismus in ChR2 sein. Um den Natriumpumpmechanismus von Krokinobacter eikastus rhodopsin 2 (KR2) zu untersuchen, haben wir in einer Pionierarbeit zeitaufgelöste Absorbtionspektroskopie mit Röntgenkristallographie kombiniert. Die Benutzung eines Quantumkaskadenlasers (QCL) ermöglichte es uns sicher zu stellen, dass kristallines KR2 vergleichbare Reaktionskinetiken aufweist als in Detergens gelost. Wir konnten daraufhin eine im Vorfeld postulierte vorübergehende Natriumbindungsstelle während des O Intermediats zwischen den Seitenketten von N112 und D251 identifizieren. Die Ergebnisse über den Ionentransportmechanismus werden die Konzipierung von Proteinvarianten für eine optogenetische Anwendung erleichtern. Bistabile G-Protein-gekoppelte Rezeptoren (GPCRs) haben zwei thermisch stabile Konformationen und sind eine vielversprechende Klasse von Rhodopsinen für einen optogentischen Schalter. Wir konnten eine erste biophysikalische Charakterisierung von jumping spider rhodopsin-1 (JSR1) durchführen. Wir schlagen, basierend auf spektroskopischen Ergebnissen, ein Model einer zwei-Photonen Reaktion vor. Während dieser Reaktionen bleibt die SB protoniert. Dies impliziert, dass eine Deprotonierung keine Voraussetzung für die Funktion von bistabilen GPCRs ist. Ein angenommenes Wassermolekül als Teil des Konterionennetzwerks konnte mit Raman Spektroskopie detektiert werden, was später durch eine Röntgenstruktur bestätigt wurde. Zusammenfassend bietet diese Arbeit Einblicke in die Mechanismen von etablierten sowie neuen optogenetischen Werkzeugen. Die Resultate werden dazu beitragen, ihre biophysikalischen Eigenschaften an die Erfordernisse der Anwendung anzupassen

Cornell Confronts the End of Mandatory Retirement

[Excerpt] In July 1995, the first author of this paper was appointed vice president of academic programs, planning and budgeting at Cornell and, at his initiative, a joint faculty-administrative committee was subsequently established, with him as chair, to look into how the university should respond to the elimination of mandatory retirement. In this chapter, we discuss the environment in which the university found itself when the committee was established, the recommendations of the committee, faculty reactions to the recommendations, and the actions that the university ultimately decided to pursue

The two-photon reversible reaction of the bistable jumping spider rhodopsin-1

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography

Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E. coli rrn Operons

AbstractPrevious electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation

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

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins

Protein conformational changes and protonation dynamics probed by a single shot using quantum-cascade-laser-based IR spectroscopy

Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations

Process and Drying Behavior Toward Higher Drying Rates of Hard Carbon Anodes for Sodium‐Ion Batteries with Different Particle Sizes: An Experimental Study in Comparison to Graphite for Lithium‐Ion‐Batteries

Sodium-ion batteries are considered to be one of the most promising postlithium batteries on the verge of commercialization. The electrode processing is expected to be similar to lithium-ion batteries. However, the producibility and material processing challenges of potential electrode materials for anodes and cathodes are poorly understood. For industrial electrode production, a deep understanding of the processing of electrode materials with different particle morphologies is of great importance. In particular, the correlation between the process conditions and the electrode properties needs to be investigated further to understand the complex interactions between the battery slurry materials, the binder system, the drying process, and the microstructure formation. One promising anode material is hard carbon. The water-based processing of hard carbon slurries presented in this article shows that the drying behavior is strongly interconnected with the particle size and particle interactions in the drying electrode. This study shows that all the hard carbons investigated do not exhibit binder migration at moderate drying rates. Even at very high drying rates (9 g m−2 s−1, 12 s drying time), an increase in adhesion force of up to 39% is observed for comparatively smaller particles compared to the adhesion force at lower drying rate

Drying of Compact and Porous NCM Cathode Electrodes in Different Multilayer Architectures: Influence of Layer Configuration and Drying Rate on Electrode Properties

Porous, nanostructured particles ensure the wetting of electrolyte up to the particle core and shortened diffusion paths, which is relevant not only for lithium-ion batteries but also for postlithium systems like sodium-ion batteries. The porous structure leads to a high C-rate capability. However, compared to conventional compact NCM, porous NCM shows a reduced adhesion force but no or only slight negative influence on C-rate capability by binder migration at higher drying rates. Herein, a multilayer concept is used to increase the adhesion force with equal or better electrochemical performance compared to single-layer electrodes. Compact particles of high volumetric energy density and porous particles with high C-rate capability are combined in a simultaneously coated multilayer electrode. Multilayers with compact NCM toward the current collector and porous NCM with reduced binder content toward the separator side show an about 16-times higher adhesion force at lower drying rate and an about ten-times higher adhesion force at increased drying rate compared to electrodes produced of porous NCM only. The specific discharge capacity of the multilayers is increased by 88% at the lower and 67% at the higher drying rate for a discharge rate of 3C compared to a single layer with compact NCM

Synergy of cations in high entropy oxide lithium ion battery anode

High entropy oxides (HEOs) with chemically disordered multi-cation structure attract intensive interest as negative electrode materials for battery applications. The outstanding electrochemical performance has been attributed to the high-entropy stabilization and the so-called ‘cocktail effect’. However, the configurational entropy of the HEO, which is thermodynamically only metastable at room-temperature, is insufficient to drive the structural reversibility during conversion-type battery reaction, and the ‘cocktail effect’ has not been explained thus far. This work unveils the multi-cations synergy of the HEO Mg$_{0.2}$Co$_{0.2}$Ni$_{0.2}$Cu$_{0.2}$Zn$_{0.2}$O at atomic and nanoscale during electrochemical reaction and explains the ‘cocktail effect’. The more electronegative elements form an electrochemically inert 3-dimensional metallic nano-network enabling electron transport. The electrochemical inactive cation stabilizes an oxide nanophase, which is semi-coherent with the metallic phase and accommodates Li$^+$ ions. This self-assembled nanostructure enables stable cycling of micron-sized particles, which bypasses the need for nanoscale pre-modification required for conventional metal oxides in battery applications. This demonstrates elemental diversity is the key for optimizing multi-cation electrode materials