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In this thesis, the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin (NmHR) is presented. Members of the rhodopsin family, such as NmHR, are integral membrane proteins comprising seven α helices and a retinal chromophore, which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive. Through photoactivation, the retinal chromophore undergoes an isomerization reaction, which then drives conformational changes in the protein. Through variations in residue composition, rhodopsin can catalyze diverse chemical reactions, including pumping protons, sodium, or chloride ions. Chloride transport is a fundamental process in biology and crucial for maintaining the electrochemical balance of the cell. The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR. Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated. This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods.

During my PhD studies at the ETH Zurich and the Paul Scherrer Institute I worked in the field of time-resolved serial femtosecond X-ray crystallography (TR-SFX), an emerging structural biology method that is performed at X-ray free-electron lasers (XFELs). The first advantage of XFELs is their provision of ultrafast 10-100 fs pulses, which allow radiation damage effects to be avoided, and obtaining structures from data collected at room temperature. The second advantage of XFELs comes upon installing a pump laser that induces protein conformational changes and allows obtaining crystallographic datasets at different times after protein activation. The protein that was subject to the study, Krokinobacter eikastus rhodopsin 2 (KR2), is the first discovered light-driven sodium pump. It was intriguing to elucidate the protein’s new sodium-pumping mechanism. It had been long debated if such a non-H+ cation pump could exist, because Na+ was supposed to be electrostatically repulsed from a positively charged retinal chromophore Schiff base. Due to the fact that outward pumping of Na+ silences neuronal signalling, KR2 is considered a next-generation tool for optogenetics.

We report on the application of a short working distance von Hamos geometry spectrometer to measure the inelastic X-ray scattering (IXS) signals from solids and liquids. In contrast to typical IXS instruments where the spectrometer geometry is fixed and the incoming beam energy is scanned, the von Hamos geometry allows measurements to be made using a fixed optical arrangement with no moving parts. Thanks to the shot-to-shot capability of the spectrometer setup, we anticipate its application for the IXS technique at X-ray free electron lasers (XFELs). We discuss the capability of the spectrometer setup for IXS studies in terms of efficiency and required total incident photon flux for a given signal-to-noise ratio. The ultimate energy resolution of the spectrometer, which is a key parameter for IXS studies, was measured to the level of 150 meV at short crystal radius thanks to the application of segmented crystals for X-ray diffraction. The short working distance is a key parameter for spectrometer efficiency that is necessary to measure weak IXS signals.

The SwissFEL X-ray Free Electron Laser (XFEL) facility started construction at the Paul Scherrer Institute (Villigen, Switzerland) in 2013 and will be ready to accept its first users in 2018 on the Aramis hard X-ray branch. In the following sections we will summarize the various aspects of the project, including the design of the soft and hard X-ray branches of the accelerator, the results of SwissFEL performance simulations, details of the photon beamlines and experimental stations, and our first commissioning results.

X-ray techniques have long been applied to chemical research, ranging from powder diffraction tools to analyse material structure to X-ray fluorescence measurements for sample composition. The development of high-brightness, accelerator-based X-ray sources has allowed chemists to use similar techniques but on more demanding samples and using more photon-hungry methods. X-ray Free Electron Lasers (XFELs) are the latest in the development of these large-scale user facilities, opening up new avenues of research and the possibility of more advanced applications for a range of research. The SwissFEL XFEL project at the Paul Scherrer Institute will begin user operation in the hard X-ray (2.1–12.4 keV) photon energy range in 2018 with soft X-ray (240–1930 eV) user operation to follow and here we will present the details of this project, it's operating capabilities, and some aspects of the experimental stations that will be particularly attractive for chemistry research. SwissFEL is a revolutionary new machine that will complement and extend the time-resolved chemistry efforts in the Swiss research community.

In preparation for picosecond pump-probe experiments at the SwissFEL X-ray laser facility, the feasibility of collectively initiating surface chemical reactions using energetic pulses of terahertz radiation is being tested. © Schweizerische Chemische Gesellschaft.

Powerful THz radiation confined in one field period or less is an adequate tool for triggering nonlinear actions. We show results towards the realization of a tunable high-power THz source based on a laser-driven frequency conversion scheme in plasma and nonlinear crystals. A powerful THz source in combination with the future X-ray Free Electron Laser facility in Switzerland (SwissFEL) holds promise for exciting experiments in a variety of different research areas.

The SwissFEL (Swiss Free Electron Laser) is a free electron laser which is being built at the Paul Scherrer Institute. The SwissFEL will be a user facility to study processes on unexplored scales of space and time. In order to achieve these aims, X-ray pulses on the order of femtoseconds must be generated. For such short X-ray pulses, the electron bunch length must be comparable to the final X-ray pulse length. In order to achieve such a short electron bunch length, it must be monitored at every compression stage. This work proposes two novel methods to measure such electron bunches, namely a novel spectral method that analyses the frequency content of electron bunches that becomes easier with shorter bunch length and a transverse deflecting cavity method which can conceptually measure a zero bunch length. Because the SwissFEL was not built at the time of the PhD, all experiments were performed at the SwissFEL Injector Test Facility (SITF). The SITF is a 200 MeV test bed linear electron accelerator. Theoretically the bunch length is limited due to the uncorrelated energy spread and the higher order components of the bunch compressor. However, due to the limited electron energy in combination with the short electron bunches, space charge effects dominate the beam in the transverse phase space as well as in the longitudinal phase space. A transverse deflecting cavity (TDC) measurement was used to measure a reference bunch length to which the spectral fluctuations could be correlated. In a TDC the electric fields are oriented perpendicular to the beam direction and streaks the head and tail in opposite directions onto a screen. In a conventional TDC measurement, the projected beam size onto the temporal axis is the current profile, provided that the intrinsic beam size is small. However, in the SITF the intrinsic beam size was large due to the aforementioned space charge effects. This led to the development of a novel bunch length analysis method in a space charge dominated beam. In the proposed analysis method, a dispersive section in combination with a TDC was used to visualise the longitudinal phase space. The longitudinal phase space was sliced in the momentum domain to reveal the difference between the intrinsic and the streaked beam in a momentum slice. The bunch length could be extracted by recording the times for all slices. Both the simulations and experiments suggest the method enables the measurement of bunch length in a space charge dominated beam in which the transverse intrinsic beam size is large. The measured minimal bunch length from simulations and experiments are of the same order, 19.5 fs to 24.5 fs, respectively. Since the TDC at PSI has a specified resolution of 20 fs at 200 pC, this method may be used to reduce the resolution. The spectral content of femtosecond electron bunches contains electron bunch length information. As an electron beam collides with an optical transition radiation (OTR) foil, radiation is emitted. The radiation is collected by means of a mirror setup into an optical fibre and into an optical spectrometer. The spectral range of the spectrometer is 316 THz (950 nm) to 1364 THz (220 nm). From OTR theory a flat OTR spectrum was expected and measured. As the electron bunch length was reduced below 70 fs, periodic fluctuations started to appear on the spectra. These fluctuations broaden with decreasing bunch length and show a good agreement with the bunch length measured with the TDC.