Sergio Carbajo

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography.

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

Laser-Induced Linear-Field Particle Acceleration in Free Space

Linear-field particle acceleration in free space (which is distinct from geometries like the linac that requires components in the vicinity of the particle) has been studied for over 20 years, and its ability to eventually produce high-quality, high energy multi-particle bunches has remained a subject of great interest. Arguments can certainly be made that linear-field particle acceleration in free space is very doubtful given that first-order electron-photon interactions are forbidden in free space. Nevertheless, we chose to develop an accurate and truly predictive theoretical formalism to explore this remote possibility when intense, few-cycle electromagnetic pulses are used in a computational experiment. The formalism includes exact treatment of Maxwell’s equations and exact treatment of the interaction among the multiple individual particles at near and far field. Several surprising results emerge. We find that electrons interacting with intense laser pulses in free space are capable of gaining substantial amounts of energy that scale linearly with the field amplitude. For example, 30u2009keV electrons (2.5% energy spread) are accelerated to 61u2009MeV (0.5% spread) and to 205u2009MeV (0.25% spread) using 250 mJ and 2.5u2009J lasers respectively. These findings carry important implications for our understanding of ultrafast electron-photon interactions in strong fields.

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

Look fast Organisms from bacteria to humans sense and react to light. Proteins that contain the light-sensitive molecule retinal couple absorption of light to conformational changes that produce a signal or move ions across a membrane. Nogly et al. used an x-ray laser to probe the earliest structural changes to the retinal chromophore within microcrystals of the ion pump bacteriorhodopsin (see the Perspective by Moffat). The excited-state retinal wiggles but is held in place so that only one double bond of retinal is capable of isomerizing. A water molecule adjacent to the proton-pumping Schiff base responds to changes in charge distribution in the chromophore even before the movement of atoms begins. Science, this issue p. eaat0094; see also p. 127 Ultrafast crystallography captures the response of the pigment of bacteriorhodopsin to absorption of light. INTRODUCTION Retinal is a light-sensitive protein ligand that is used by all domains of life to process the information and energy content of light. Retinal-binding proteins are integral membrane proteins that drive vital biological processes, including light sensing for spatial orientation and circadian clock adjustment, as well as maintaining electrochemical gradients through ion transport. They also form the basis for optogenetic manipulation of neural cells. How the protein environment guides retinal isomerization on a subpicosecond time scale toward a single high-yield product is a fundamental outstanding question in photobiology. RATIONALE Light-induced isomerization of retinal is among the fastest reactions known in biology. It has been widely studied by spectroscopic techniques to probe the evolution of spectral intermediates over time. Using x-ray free-electron lasers (XFELs), it is now possible to observe ultrafast photochemical reactions and their induced molecular motions within proteins on scales of femtoseconds to milliseconds with near-atomic structural resolution. In this work, we used XFEL radiation to study the structural dynamics of retinal isomerization in the light-driven proton-pump bacteriorhodopsin (bR). The principal mechanism of isomerization in this prototypical retinal-binding protein has direct relevance for all other members of this important family of membrane proteins, and it provides insight into how protein environments catalyze photochemical reactions in general. RESULTS We collected high-resolution x-ray diffraction data from bR microcrystals injected across the femtosecond x-ray pulses of the Linac Coherent Light Source after excitation of the retinal chromophore by an optical laser pulse. X-ray diffraction images were sorted into temporal subgroups with a precision of about 200 fs. A series of 18 overlapping difference Fourier electron density maps reveal structural changes over the first picosecond of retinal photoexcitation. Complementary data for time delays of 10 ps and 8.33 ms allow us to resolve the later stages of the reaction. In combination with refined crystallographic structures at pump-probe delays corresponding to where the spectroscopically characterized I, J, K, and M intermediates form in solution, our time-resolved structural data reveal the trajectory of retinal isomerization and provide atomic details at key points along the reaction. The aspartic acid residues of the retinal counterion and functional water molecules in close proximity to the retinal Schiff base respond collectively to the formation and decay of the excited state. This collective motion sets the stage for retinal isomerization, which proceeds via a twisted retinal configuration. Quantum mechanics/molecular mechanics simulations provide theoretical support for this structural evolution. CONCLUSION Our observations reveal how, concomitant with the formation of the earliest excited state, the retinal-binding pocket opens up in close proximity to the isomerizing bond. We propose that ultrafast charge transfer along retinal is a driving force for collective motions that contribute to the stereoselectivity and efficiency of retinal isomerization within a protein scaffold. Vibrational quake-like motions extending from retinal to the protein may also be a mechanism through which excess energy is released in a nonradiative fashion. Time-resolved serial crystallography resolves ultrafast atomic motions of retinal and the surrounding protein following photoexcitation. Retinal evolves from an all-trans conformation in the ground state toward a twisted 13-cis retinal over the course of a few hundred femtoseconds. The complex counterion, formed by two aspartic acid residues (Asp) and a water molecule (Wat), responds to changes in the electronic structure of the chromophore on the same time scale as the formation of the excited state. 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.

Laser power meters as an X-ray power diagnostic for LCLS-II

Laser power meters are being developed as a compact X-ray power diagnostic for LCLS-II and are characterized for their responsivity, linearity and vacuum compatibility. The power meters are calibrated against X-ray photodiodes and a gas monitor detector.