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

Ultrafast protein response in the Pfr state of Cph1 phytochrome

Photoisomerization is a fundamental process in several classes of photoreceptors. Phytochromes sense red and far-red light in their Pr and Pfr states, respectively. Upon light absorption, these states react via individual photoreactions to the other state. Cph1 phytochrome shows a photoisomerization of its phycocyanobilin (PCB) chromophore in the Pfr state with a time constant of 0.7 ps. The dynamics of the PCB chromophore has been described, but whether or not the apoprotein exhibits an ultrafast response too, is not known. Here, we compare the photoreaction of 13C/15N labeled apoprotein with unlabeled apoprotein to unravel ultrafast apoprotein dynamics in Cph1. In the spectral range from 1750 to 1620 cm−1 we assigned several signals due to ultrafast apoprotein dynamics. A bleaching signal at 1724 cm−1 is tentatively assigned to deprotonation of a carboxylic acid, probably Asp207, and signals around 1670 cm−1 are assigned to amide I vibrations of the capping helix close to the chromophore. These signals remain after photoisomerization. The apoprotein dynamics appear upon photoexcitation or concomitant with chromophore isomerization. Thus, apoprotein dynamics occur prior to and after photoisomerization on an ultrafast time-scale. We discuss the origin of the ultrafast apoprotein response with the ‘Coulomb hammer’ mechanism, i.e. an impulsive change of electric field and Coulombic force around the chromophore upon excitation

A Combined Vis-Pump Supercontinuum Probe and Broadband Fluorescence Up- Conversion Study

Corroles are a developing class of tetrapyrrole-based molecules with significant chemical potential and relatively unexplored photophysical properties. We combined femtosecond broadband fluorescence up-conversion and fs broadband Vis-pump Vis-probe spectroscopy to comprehensively characterize the photoreaction of 5,10,15-tris-pentafluorophenyl-corrolato-antimony(V )-trans-difluoride (Sb-tpfc-F2). Upon fs Soret band excitation at ~400 nm, the energy relaxed almost completely to Q band electronic excited states with a time constant of 500 ± 100 fs; this is evident from the decay of Soret band fluorescence at around 430 nm and the rise time of Q band fluorescence, as well as from Q band stimulated emission signals at 600 and 650 nm with the same time constant. Relaxation processes on a time scale of 10 and 20 ps were observed in the fluorescence and absorption signals. Triplet formation showed a time constant of 400 ps, with an intersystem crossing yield from the Q band to the triplet manifold of between 95% and 99%. This efficient triplet formation is due to the spin-orbit coupling of the antimony ion

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

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore’s pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes

Femtosekunden Spektroskopie an Corrolen, Phytochromen, Channelrhodopsinen und Grundzustandsreaktionen

Photoreactions are ubiquitous and the foundation of life. The outcome of a photoreaction is mostly determined by its first steps, which happen within few picoseconds. Therefore the study of the initial part of a photoreaction is indispensable for its full understanding. This thesis presents the results of femtosecond pump-probe spectroscopy, the tool of choice for observing photoreactions on an ultrashort timescale, on four different systems: Corrole, Channelrhodopsin 1, Cph1-Phytochrome and a bimolecular ground-state reaction. Corroles are cyclic tetra-pyrroles similar to porphyrins. They show promising characteristics as photosensitizers in photodynamic therapy for cancer. Here, a high triplet yield is crucial. My results show that the addition of bromine to the macrocycle induces efficient intersystem crossing on a 100 ps timescale with a yield near unity. Observation of the ISC in the mid-infrared reveals a distinct spectrum of the triplet state, usable as a marker for the spin-state. Phytochromes are an omnipresent class of photoreceptor proteins initially found in plants. Phytochromes have two semi-stable states, the red and the far-red absorbing state. Recent studies show that these states itself are heterogeneous, but whether this heterogeneity ifluences the photoreaction was still unknown. Using Vis-pump IR/Vis-probe spectroscopy, we show that this is the case. The transient spectra expose two diffferent bleaching bands of the ring-D carboxyl stretching vibration, which we assign to sub-states with diffferent ring-D orientations. Since these two bands show different transient changes, we conclude that the heterogeneity indeed influences the photoreaction. Channelrhodopsin1 is a transmembrane protein which functions as a light-activated cation channel. Channelrhodopsins variants are the primary tools in the new field of Optogenetics. Visible pump visible probe spectroscopy reveals an unusually fast 100 fs all-trans to 13-cis photo-isomerization of the retinal photoreceptor and excitation wavelength dependent dynamics. In the mid-IR fingerprint regi- on,we find evidence that the isomerization outcome depends on the conformation of the ground-state. Using polarisation resolved Vis-pump IR-probe spectroscopy, we assign several priorly unassigned bands in the amide region. Comparison of our results with the C1C2-chimaera- and a homology-structure suggests that ultrafast appearing amide I band, which is strong in CaChR1,has its origin in one of the tryptophans belonging to the retinal cage. Moreover, my data excludes the existence of protonated carboxylic groups coupled to the retinal in ground-state, since no corresponding bands are observed. Bimolecular ground-state reactions are the most common reactions in chemistry. They can be described by transition state theory, in which the reaction is described by a potential energy surface. The initial and the product state are minima separated by a barrier and the shortest path over the barri- er only depends on a subset of coordinates, called reaction coordinates. Since they can be represented by normal modes, it is generally believed that IR-excitation of the correct modes can initiate the reaction. However, this idea was not yet experimentally proven. To find the missing proof and make selective laser-chemistry possible, we applied fs IR-pump IR-probe spectroscopy to a mixture of cyclohexanol and isocyanate, which react to Cyclohexyl-carbanilate at room temperature. We observe the rise of vibrational bands belonging to the product and the rise of bleaching bands belonging to the reagents on a 10 ps scale, which shows that the reaction is initiated by the IR-excitation. We support this finding by comparing the reaction rates with and without IR-illumination, showing a 24 % increase. This finding offers entirely new ways to optimize low yield reactions

2D IR raw data from MeSCN in water

Full data from a measurement run using a Phasetech 2D-spectrometer powered by a HE Topas using the orignal QuickControl software. The sample is MeSCN, an commonly used test sample with a rather long virbational lifetime. Reupload since the inital zip archive used a windows only deflate algorithm.</p

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 conductivity

Ultrafast Dynamics of Sb-Corroles: A Combined Vis-Pump Supercontinuum Probe and Broadband Fluorescence Up-Conversion Study

Corroles are a developing class of tetrapyrrole-based molecules with significant chemical potential and relatively unexplored photophysical properties. We combined femtosecond broadband fluorescence up-conversion and fs broadband Vis-pump Vis-probe spectroscopy to comprehensively characterize the photoreaction of 5,10,15-tris-pentafluorophenyl-corrolato-antimony(V)-trans-difluoride (Sb-tpfc-F2). Upon fs Soret band excitation at ~400 nm, the energy relaxed almost completely to Q band electronic excited states with a time constant of 500 ± 100 fs; this is evident from the decay of Soret band fluorescence at around 430 nm and the rise time of Q band fluorescence, as well as from Q band stimulated emission signals at 600 and 650 nm with the same time constant. Relaxation processes on a time scale of 10 and 20 ps were observed in the fluorescence and absorption signals. Triplet formation showed a time constant of 400 ps, with an intersystem crossing yield from the Q band to the triplet manifold of between 95% and 99%. This efficient triplet formation is due to the spin-orbit coupling of the antimony ion.Peer Reviewe