Cellular lipid membranes can – and often do – support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches. I introduce the microbial rhodopsin Archaerhodopsin-3 (Arch), a transmembrane protein from Halorubrum sodomense. The fluorescence of Arch is a function of membrane potential, allowing it to serve as an optical voltage reporter. We use time-dependent pump-probe spectroscopy to interrogate the light- and voltage- dependent conformational dynamics of this protein, to elucidate the mechanism of voltage-dependent fluorescence in Arch. I then present two new methods for imaging voltage using engineered variants of Arch. Both techniques take advantage of the unique photophysical properties of Arch(D95X) mutants. The first method, Flash Memory, records a photochemical imprint of the activity state -- firing or not firing -- of a neuron at a user-selected moment in time. The Flash Memory technique decouples the recording of neural activity from its readout, and can potentially allow us to take large-scale snapshots of voltage (e.g. maps of activity in a whole mouse brain). The second method allows for the quantitative optical measurement of membrane potential. This technique overcomes the problems that typically hinder intensity-based measurements by encoding a measurement of voltage in the time domain. Finally, I present a method to visualize cellular responses to changes in membrane potential. I engineer mutants of Channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii that is used for optical control of neural activity, and use these optogenetic actuators in conjunction with GFP-based sensors to study the activity-dependent behavior of cultured neurons.

La photopolymérisation est une technologie qui gagne de plus en plus d’importance de par ses nombreuses applications et ses énormes avantages par rapport à la polymérisation thermique tels que le respect de l’environnement, des coûts économiques maitrisés car la technologie est mise en œuvre à température ambiante et ne requiert qu’une faible consommation d'énergie. De plus, elle n'utilise pas ou très peu de solvants, d'où la réduction d’émission de produits polluants. Le processus de polymérisation photochimique présente également l’avantage d’être très rapide : en effet les réactions de photopolymérisation sont souvent rapides voire même quasi instantanées. De plus le procédé de polymérisation devient photolatent : la polymérisation impliquant les formulations actives seulement en présence de lumière, la réaction peut être déclenchée « quasi à la demande ». Ce processus chimique nécessite des composants nommés « photoamorceurs », lesquels absorbent la lumière et produisent le démarrage de la réaction de polymérisation. En particulier, le développement de systèmes photoamorceurs capables d’absorber de la lumière visible a un intérêt croissant pour différentes applications industrielles, notamment l’holographie. Dans la cadre de cette thèse, des diverses systèmes photoamorceurs contenant un colorant et un ou deux co-amorceurs, qui après réaction avec les états excités des colorants génèrent les radicaux actifs, ont été étudiés, depuis leurs propriétés photophysiques fondamentales et moléculaires, jusqu’aux applications et performances pour la polymérisation des résines acrylates. Cette thèse est articulée autour de six chapitres. Dans le premier chapitre une introduction et une étude bibliographique des différents systèmes photoamorceurs développés ces dernières années sont présentées et comparés. Les chapitres deux et trois sont consacrés à l’étude des propriétés photochimiques et photophysiques réalisés sur des photoamorceurs absorbant de la lumière ultraviolette et visible respectivement. Les techniques utilisés dans ces chapitres incluent, mais ne sont pas limités à, la spectroscopie d’absorption UV-Vis stationnaire, la fluorescence, la photolyse éclaire (LFP), la fluorescence résolue en temps par comptage de photon unique (TC-SPC), la spectroscopie ultrarapide nanoseconde et femtoseconde, la spectroscopie de résonance paramagnétique électronique (EPR), entre autres. Le chapitre deux porte sur l’étude de la photophysique d’un colorant de type cyanine : l’astrazone Orange R (AO R). La photophysique de ce colorant n’est pas connue et une étude exhaustive a été menée avec des spectroscopies ultra rapides (femtoseconde) ainsi que par modélisation moléculaire. Malgré sa photophysique compliquée ce colorant, fonctionne comme un photoamorceur très efficace dans la région bleue du spectre électromagnétique. Les chapitres suivants forment une deuxième partie de la thèse dédiée à l’étude des mécanismes d’amorçage de photopolymérisation, dont le chapitre quatre aborde le cas d’irradiation le plus « traditionnel », qui est l’irradiation en mode continu (ou CW). Est ensuite abordé l’étude de la polymérisation sous irradiation pulsée ultra-courte : dans cette partie la source d’irradiation continue classique est remplacée par un laser Nd :Yag qui produit des impulsions lumineuses d’une durée de quelques 9-10 nanosecondes. Cette polymérisation avec laser pulsé ou PLP est très originale et sera étudiée par spectroscopie infrarouge à transformée de Fourier résolue en temps (RT-FTIR). Dans le chapitre trois, donc, on retrouve les études réalisés avec différents types de systèmes photoamorceurs visibles. Ainsi, les propriétés des colorants capables d’absorber de la lumière visible à différentes longueurs d’onde ont été étudiées dans des systèmes photoamorceurs à deux et trois composants. / Photopolymerization is a technology that is gaining more and more importance due to its numerous applications and its advantages compared to thermic polymerization. This chemical process requires compounds called photoinitiators, which absorb light and produce the initiation of the radical polymerization. The development of photoinitiating systems (PIS) which are able to absorb visible light have an increasing interest due to its industrial applications, such as holographic recording. During this thesis, many PIS were studied, from its photophysical properties to its application in acrylate polymerization. There are many different types of photoinitiators. The most classical ones are Type I PI, which are molecules that overcome homolytic cleavage from their excited state, generating initiating radicals immediately after photon absorption. Type II PI, in contrast, are composed by two molecules: one that absorbs the photon, and other that will react with the excited state of the first via electron transfer or hydrogen transfer, generating radicals that will be able to initiate polymerization. In last place, there are the most efficient Photocyclic Initiating Systems (PCIS) whose mechanism is more complicated and will be widely discussed within these pages. A state of the art of the PIS available up to date is made in the first chapter. Given the importance of the properties of the molecules involved in the photoinitiating process, the studies of the photophysical properties of a Photoinitiator, the Astrazone Orange (AO), are shown. It was found that this molecule suffers an isomerization process from its excited state, which then comes slowly back to the more stable conformer. This process being viscosity-dependent makes AO a suitable photoinitiator for polymerization with visible light in highly viscous media. The last three chapters of this thesis are devoted to the study of a novel technique called Pulsed Laser Polymerization (PLP). This technique consists in the irradiation of the samples with a short duration pulsed laser, which allows the separation of the initiation steps of the polymerization reaction from the steps of propagation and termination. A simple Type I PI was used as a model to study the properties of this technique of polymerization. The monomer conversion was registered by RT-FTIR and analyzed. To a better understanding of these results, a mathematical model was developed. Thanks to it, it was possible to collect valuable information about propagation and termination rate constants (kp and kt, respectively), the variation of viscosity with conversion and other aspects relatives to PLP mechanism. Furthermore, the efficiency of many visible light PIS was studied by PLP. Their performance was compared and studied and contrasted with the classical continuous irradiation mode (CW). The characteristics that a PIS must have in order to show efficient polymerization in PLP mode were found and discussed. Finally, the effect of formulation viscosity in PLP and CW was analyzed by diluting the sample with different amounts of DMSO. In PLP, it was seen that the highest conversion is found for the most concentrated samples, while the opposite effect is noticed in CW. This result is attributed to the different conditions given by the difference in irradiation methods.

"An apparatus for the delivery of radiation to the tip-sample interface of an Atomic Force Microscope (AFM) is demonstrated. The Pulsed Light Delivery System (PLDS) was fabricated to probe photoinduced conformational changes of molecules using an AFM. The PLDS is 67 mm long, 59 mm wide, and 21 mm high, leaving clearance to mount the PLDS and a microscope slide coated with a thin film of photoactive molecules beneath the cantilever tip of a stand-alone AFM. The PLDS is coupled into a fiber pigtailed Nd:Yag frequency doubled laser, operating at a wavelength of 532 nm. The radiation delivered to a sample through the PLDS can be configured for continuous or pulsed mode. The maximum continuous wave (CW) power delivered was 0.903 mW and the minimum pulse width was 12.3 ms (maximal 401 ms), corresponding to a minimal energy of 0.150 nJ (maximal 362 nJ), and had a cycle duration of 10.0 ms. The PLDS consists of micro-optical components 3.0 mm and smaller in diameter. The optical design was inspired by the three-beam pick-up method used in CD players, which could provide a method to focus the pulse of light onto the sample layer. In addition, the system can be easily modified for different operational parameters (pulse width, wavelength, and power). As proof that the prototype design works, we observed a photoinduced ‘bimetallic’ bending of the cantilever, as evidenced by observing no photoinduced bending when a reflective-coated cantilever was replaced by an uncoated cantilever. Using the apparatus will allow investigation of many different types of molecules exhibiting photoinduced isomerization."

Kanalrhodopsine (ChRs) sind lichtgesteuerte Ionenkanäle, welche nach Absorption eines Photons durch den Retinal-Cofaktor einen passiven Ionentransport über die Zellmembran katalysieren. Im Zuge von optogenetischen Anwendungen wird diese Reaktion für die Beeinflussung der Ionenhomöostase von verschiedenen Zelltypen und Geweben ausgenutzt. Zu Beginn dieser Arbeit wurden lichtinduzierte Strukturänderungen und Protontransferschritte in einem breiten Zeitbereich (Nanosekunden bis Minuten) in dem grün-absorbierenden ChR ReaChR mithilfe von stationärer und transienter UV-vis- und Fourier-Transform-Infrarot-Spektroskopie (FTIR) untersucht. Auf Basis der experimentellen Daten wurde ein komplexes Photozyklus-Modell konzipiert. Anschließend wurde die IR-aktive, nichtkanonische Aminosäure p-Azido-L-phenylalanin (azF) mittels Stopp-Codon-Suppression ortsspezifisch an mehreren Positionen innerhalb der vermuteten ionenleitenden Kanalpore in ReaChR inkorporiert und mit FTIR untersucht. azF ist sensitiv gegenüber Polaritätsänderungen und absorbiert in einem hochfrequenten Bereich (~2100 cm-1). Aufgrund der großen spektralen Separation zu endogenen Proteinschwingungen (< 1800 cm-1) können globale Konformations- und lokale Hydratisierungsänderungen simultan detektiert werden. Die erhobenen Daten leisten einen wichtigen Beitrag zum Verständnis der Bildung einer temporären Wasserpore in ChRs und demonstrieren zum ersten Mal den erfolgreichen in-vivo-Einbau einer artifiziellen Aminosäure in mikrobielle Rhodopsine und dessen schwingungsspektroskopische Analyse. Die Methode bietet aufgrund ihrer hohen Ortsauflösung ein großes Potential für die Studie von Mikroumgebungen innerhalb komplexer Proteinensemble. / Channelrhodopsins (ChRs) are light-gated ion channels. Upon absorption of a photon, the retinal chromophore isomerizes and drives conformational changes within the protein, which lead to a passive ion transport across the cell membrane. This capability is used for optogenetic applications to manipulate ionic homeostasis of different cell types and entire organisms. Within the work, light-induced structural changes and proton transfer steps were studied in the green-absorbing ChR ReaChR in great detail by steady-state and transient UV-vis and Fourier transform infrared spectroscopy (FTIR). The data were merged into a complex photocycle model. Next, the IR-active, unnatural amino acid p-azido-L-phenylalanine (azF) was site-specifically introduced at several sites of the putative ion pore of ReaChR by stop codon suppression. azF is sensitive to polarity changes and absorbs in a clear spectral window lacking endogenous protein vibrations. Thus, FTIR measurements of labeled mutants report for global conformational changes (< 1800 cm-1) and local hydration changes (~2100 cm-1) simultaneously. The presented findings reveal crucial insights regarding formation of a transient water pore in ChRs and demonstrate the first report of the successful in-vivo incorporation of an artificial amino acid into a microbial rhodopsin and its subsequent spectroscopic investigation. Additionally, the so far unprecedented spatial resolution renders this methodology superior over conventional FTIR methods to study microenvironments within complex protein ensembles.

Histidinkinase-Rhodopsine (HKRs) können als besondere Gruppe der Hybrid-Histidinkinasen beschrieben werden, deren N-terminale sensorische Domäne ein mikrobielles Rhodopsin ist. HKR-codierende Sequenzen konnten in den Genomen verschiedener Algen, Pilze und Amoeben gefunden werden doch ihre Aufgaben und Wirkungsweisen sind bisher ungeklärt. Im Rahmen dieser Arbeit wurden die rekombinanten Rhodopsin-Domänen von zwei HKRs mit verschiedenen spektroskopischen Techniken charakterisiert. Sie zeigten mehrere Besonderheiten. Das Rhodopsin-Fragment von Cr-HKR1 aus Chlamydomonas reinhardtii kann durch alternierende kurzwellige und langwellige Belichtung zwischen zwei stabilen Absorptionsformen konvertiert werden: einer Blaulicht-absorbierenden (Rh-Bl) und einer UVA-Licht-absorbierenden Form (Rh-UV). Dies resultiert aus der ungewöhnlichen thermischen Stabilität des Zustandes mit deprotonierter Schiff’scher Base. Das zweite charakterisierte HKR, die Os-HKR-Rhodopsin-Domäne aus der marinen Picoalge Ostreococcus tauri, zeigt eine Dunkelabsorption von 505 nm. Auch Os-HKR ist photochrom und die deprotonierte Spezies kann effizient akkumuliert werden. Diese P400-Absorptionsform ist jedoch nicht völlig stabil sondern es kommt nach Belichtungsende zur langsamen Dunkelzustands-Regeneration. Überraschenderweise konnte die Bindung sowie die transiente Abgabe eines Anions während des Os-HKR-Photozyklus festgestellt werden. Somit beeinflusst nicht nur das Licht, sondern auch das Salz in der Umgebung die Os-HKR-Reaktionen. Aufgrund ihrer photochromen Eigenschaften werden die HKRs als wirksame lichtinduzierte Schalter für die C-terminalen Signaltransduktionsdomänen postuliert. Schwingungsspektroskopische Analysen deckten eine Heterogenität hinsichtlich der im Protein gebundenen Retinal‐Konfiguration sowie die Existenz von zwei parallelen Photozyklen auf. Jeder dieser Photozyklen geht aus einer der beiden Retinal-Isomere hervor. / Histidine kinase rhodopsins (HKRs) can be described as hybrid histidine kinases with a microbial rhodopsin as N-terminal sensory domain. HKR-encoding sequences were found in the genomes of various unicellular organisms such as algae, fungi and amoeba but their mechanistic and physiologic function is unknown. During this work the absorptive properties of the recombinant rhodopsin domains of two HKRs were studied by the usage of different spectroscopic techniques. Both HKRs showed unusual characteristics. The rhodopsin fragment of Cr‐HKR1 from Chlamydomonas reinhardtii can be interconverted between two stable absorbance forms by the alternate application of short‐ and long‐wavelength light: a blue light-absorbing dark form (Rh-Bl) and a UVA light-absorbing form (Rh-UV). This unusual photocycle results from the uncommon thermal stability of the absorbance state with a deprotonated retinal Schiff base. The second studied HKR, the Os‐HKR rhodopsin domain from the marine picoalga Ostreococcus tauri, shows an absorbance maximum at 505 nm in darkness. Likewise Cr‐HKR1 the Os‐HKR is photochromic and the deprotonated form P400 can be efficiently accumulated. But the Os-HKR P400-form is not completely stable. A slow dark state recovery occurs. Surprisingly the dark state absorbance of Os‐HKR was found to be dependent on anion binding in the protein. Furthermore during the photocycle the transient anion release occurs and therefore not only light but also salt impacts the Os-HKR-reactions. Due to their pronounced photochromic properties, the HKRs are postulated to act as effective molecular switches for the C-terminal signal transduction domains in response to the light conditions. Vibrational spectroscopy revealed the heterogeneity with regard to the retinal configuration bound in the HKRs suggesting the existence of two parallel photocycles. Either of these photocycles originates from one of the two retinal isoforms.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet. / Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.