A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the ...photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.
The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast ...structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent ...pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
When a molecule interacts with light, its electrons can absorb energy from the electromagnetic field by rapidly rearranging their positions. This constitutes the first step of photochemical and ...photophysical processes that include primary events in human vision and photosynthesis. Here, we report the direct measurement of the initial redistribution of electron density when the molecule 1,3-cyclohexadiene (CHD) is optically excited. Our experiments exploit the intense, ultrashort hard x-ray pulses of the Linac Coherent Light Source (LCLS) to map the change in electron density using ultrafast x-ray scattering. The nature of the excited electronic state is identified with excellent spatial resolution and in good agreement with theoretical predictions. The excited state electron density distributions are thus amenable to direct experimental observation.
Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecuies. We used microcrystals of ...photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)
. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 ...femtoseconds to the all-trans conformation
, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature
to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II ...(PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment ...protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn
CaO
cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S
to S
), in which S
is the dark-stable state and S
is the last semi-stable state before O-O bond formation and O
evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S
), two-flash illuminated (2F; S
-enriched), and ammonia-bound two-flash illuminated (2F-NH
; S
-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S
state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn
CaO
cluster in the S
and S
states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed ...as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.
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•Crystal structure of the human Angiotensin II type 1 receptor at 2.9-Å resolution•Structure is solved by X-ray laser serial femtosecond crystallography•Antagonist ZD7155 forms critical interactions with Tyr35, Trp84 and Arg167•Docking reveals binding modes of common angiotensin receptor blockers
Structure determination of human Angiotensin II type 1 receptor bound to an antagonist using serial femtosecond crystallography with X-ray free-electron laser and docking studies of other common anti-hypertensive drugs into the structure offer insights into design of blood pressure modulators.
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.
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