Jan Davidsson

Time-resolved protein nanocrystallography using an X-ray free-electron laser

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

In vivo protein crystallization opens new routes in structural biology

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

Lipidic phase membrane protein serial femtosecond crystallography.

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

Structural Dynamics of Light-Driven Proton Pumps

Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.

A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals structural changes in bacteriorhodopsin. 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.

Light-Induced Structural Changes in a Photosynthetic Reaction Center Caught by Laue Diffraction

Light Structures Absorption of light by photosynthetic reaction centers causes structural changes and triggers a series of electron transfer reactions, resulting in a transmembrane potential difference that can be used to drive the subsequent chemistry. The initial electron transfer generates a charge-separated state that must be stabilized to prevent dissipation of energy through recombination. Wöhri et al. (p. 630) have used time-resolved Laue diffraction crystallography to observe light-induced conformational changes that occur within milliseconds of photooxidation of the dimer of bacteriochlorophyll molecules, known as the “special pair,” in the photosynthetic reaction center of Blastochloris viridis. Stabilization appears to occur because of the deprotonation of a conserved tyrosine residue that moves closer to the special pair. Fleeting molecular events are observed as light illuminates chlorophyll to initiate photosynthesis. Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

Photodissociation of aryl halides in the gas phase studied with femtosecond pump-probe spectroscopy

Abstract The photodissociation dynamics of iodo-, bromo- and chlorobenzene upon excitation at 266 nm have been studied by femtosecond pump-probe spectroscopy combined with time-of-flight (TOF) mass spectrometric detection. The kinetics are characterized by a single time constant for chlorobenzene (1 ns) and bromobenzene (28 ps), and by two time constants for iodobenzene (700 and 350 fs). The time constant of 350 fs is due to direct dissociation. The increase of the other time constants with decreasing halogen mass supports the assignment of these constants to the decay of the initially excited ( π , π ∗ ) states to the repulsive triplet ( n , σ ∗ ) state due to spin–orbit coupling.

Ultrafast Electron Transfer Dynamics of a Zn(II)porphyrin-Viologen Complex Revisited: S2 vs S1 Reactions and Survival of Excess Excitation Energy

The photoinduced electron transfer reactions in a self-assembled 1:1 complex of zinc(II)tetrasulphonatophenylporphyrin (ZnTPPS(4-)) and methylviologen (MV(2+)) in aqueous solution were investigated with transient absorption spectroscopy. ZnTPPS(4-) was excited either in the Soret or one of the two Q-bands, corresponding to excitation into the S(2) and S(1) states, respectively. The resulting electron transfer to MV(2+) occurred, surprisingly, with the same time constant of τ(FET) = 180 fs from both electronic states. The subsequent back electron transfer was rapid, and the kinetics was independent of the initially excited state (τ(BET) = 700 fs). However, ground state reactants in a set of vibrationally excited states were observed. The amount of vibrationally excited ground states detected increased with increasing energy of the initial excited state, showing that excess excitation energy survived a two-step electron transfer reaction in solution. Differences in the ZnTPSS(•3-)/MV(•+) spectra suggest that the forward electron transfer from the S(2) state at least partially produces an electronically excited charge transfer state, which effectively suppresses the influence of the inverted regime. Other possible reasons for the similar electron transfer rates for the different excited states are also discussed.

Variation of Excitation Energy Influences the Product Distribution of a Two-Step Electron Transfer: S2 vs S1 Electron Transfer in a Zn(II)porphyrin−Viologen Complex

We have, for the first time for molecular systems in solution, shown a case where the variation of excitation energy influences the product distribution of a two-step electron transfer. The photoexcitation was to the porphyrin-localized S(2) state or either of the S(1)(v = 1) or S(1)(v = 0) states of an aqueous Zn(II)-meso-tetrasulphonatophenyl-porphyrin-methylviologen (ZnTPPS(4-)/MV(2+)) complex. Both forward and back electron transfer occur on a subpicosecond time scale (tau(FET) approximately 0.2, tau(BET) = 0.7 ps). The excess energy of the higher excitations partially survives both electron transfer steps. This is seen by different distributions of unrelaxed ground states, which are generated by the back electron transfer and has unique UV-vis spectroscopic signatures. State selective electron transfer opens interesting possibilities for reaction control, and the results represent initial steps in that direction.