Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 A resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 A using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 A from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 A between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously

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Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

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.

SwissFEL: The Swiss X-ray Free Electron Laser

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

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Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytosk...

Multiscale Simulations of Biological Membranes: The Challenge To Understand Biological Phenomena in a Living Substance.

BACKGROUND Channelrhodopsins (ChRs) are naturally occurring light-gated ion channels that are important for allowing motile algal cells to find suitable light levels. In neuroscience, ChRs have become broadly significant for helping to enable the control of specific circuit elements with light (i.e., optogenetics). Research into how sensation, cognition, and behavior arise from neuronal activity dynamics has been enabled by the expression of ChRs, and other members of the microbial opsin family, in specific cells or in specific connections within nervous systems of behaving animals. Both the unique light-gated channels themselves and opportunities for their biological application have been under intense investigation. The resulting studies of atomic-scale structure-function relationships have led not only to sophisticated understanding of the underlying chemical processes governing these unique seven-transmembrane channels from the plant kingdom, but also (via optogenetics) to the discovery of fundamental neural circuit principles underlying adaptive and maladaptive behavior in animals. ADVANCES The atomic-scale understanding of light-gated ion channel function has spanned the key processes of activation/deactivation gating, light adaptation, color tuning, and ion selectivity. A ChR crystal structure–derived, molecular dynamics–calculated pore snapshot (top left panel of the figure) summarizes the wide scope of biophysical and biochemical discoveries. Molecular modeling and redesign have created multiple modes of coupling between delivered photons and spikes in an approach that has illuminated basic principles of protein function and also created new tools for optogenetics. In the top right panel of the figure, the top trace shows a photon-spike transduction mode arising from the ChETA mutation, which results in high-speed, high-fidelity single blue flash–single spike coupling. The second trace shows red photon-spike transduction arising from a redshifted ChR found in nature and then engineered for stronger, more redshifted performance (C1V1). The third trace shows bistable excitation photon-spike logic, in which step-function opsin (SFO) mutations were introduced to create stalled photocycles, allowing stable excitation without continuous light delivery. The bottom trace shows bistable inhibition photon-spike logic; ChRs that are normally cation-conducting, and are therefore excitatory in neural systems, were converted to anion-conducting (inhibitory) ChRs by replacing negatively charged pore residues, followed by SFO mutations for bistability. The C1V1 and SFO designs together allowed us to determine that the medial prefrontal neocortex modulates interactions between two distant subcortical structures to control reward-mediating physiology and behavior (clarityresourcecenter.org/ofMRI.html; www.optogenetics.org). OUTLOOK The ChR light-gated pore will continue to be studied for its own elegant properties, which are paradigmatic among ion channels because light-gated systems allow structure-function analysis on the femtosecond time scale. Meanwhile, psychiatry has already yielded some of its deepest mysteries to ChR pore structural insights, including in explorations of clinically relevant behavioral states such as anhedonia. Many more opportunities for ChRs in basic neuroscience remain untapped, with the potential for precision redesign to achieve new applications and new roles integrated with other advanced technologies.

The form and function of channelrhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

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Byeonghyun Jeon

Papers

A three-dimensional movie of structural changes in bacteriorhodopsin