Visualizing photochemical dynamics in solution through picosecond x-ray scattering.

A photoexcited state of molecular iodine in solution is observed using diffuse x-ray scattering at a synchrotron source. The measured changes in the diffuse scattering profile were consistent with earlier models of iodine's photodissociation and geminate recombination reaction, for which the recombined A/A(') state has a 0.4 A greater interatomic spacing than the resting state and has a lifetime of 500 ps in CH2Cl2. This technique should find application in the study of increasingly complicated photochemical systems which undergo structural rearrangements following rapid photolysis.

X-ray structure of sensory rhodopsin II at 2.1-A resolution.

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from Natronobacterium pharaonis (pSRII) at 2.1-A resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with HtrII. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.

Spectroscopic characterization of bacteriorhodopsin's L-intermediate in 3D crystals cooled to 170 K.

Spectra are presented from a single 3D microcrystal of bacteriorhodopsin (bR) cooled to 170 K under various illumination conditions. This set is necessary and sufficient to assign the relevant crystal reference spectra. A spectral decomposition of the difference spectrum obtained following the trapping protocol of Royant et al. (2000) (Nature 406, 645-648) is given, confirming that the low temperature L-intermediate was the species that dominated the structural rearrangements previously reported. Smaller contributions from the K and M spectral intermediates are also quantified. Mechanistic insights derived from the X-ray structures of the early bR intermediates are discussed.

Analyzing protein functions in four dimensions.

Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1 nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle.

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.

Potential for biomolecular imaging with femtosecond X-ray pulses.

Sample damage by X-rays and other radiation limits the resolution of structural studies on non-repetitive and non-reproducible structures such as individual biomolecules or cells. Cooling can slow sample deterioration, but cannot eliminate damage-induced sample movement during the time needed for conventional measurements. Analyses of the dynamics of damage formation suggest that the conventional damage barrier (about 200 X-ray photons per A2 with X-rays of 12 keV energy or 1 A wavelength) may be extended at very high dose rates and very short exposure times. Here we have used computer simulations to investigate the structural information that can be recovered from the scattering of intense femtosecond X-ray pulses by single protein molecules and small assemblies. Estimations of radiation damage as a function of photon energy, pulse length, integrated pulse intensity and sample size show that experiments using very high X-ray dose rates and ultrashort exposures may provide useful structural information before radiation damage destroys the sample. We predict that such ultrashort, high-intensity X-ray pulses from free-electron lasers that are currently under development, in combination with container-free sample handling methods based on spraying techniques, will provide a new approach to structural determinations with X-rays.

Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin.

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 A resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the protein's core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the protein's surface, aiding proton release to the extracellular medium.

Deconvoluting ultrafast structural dynamics: temporal resolution beyond the pulse length of synchrotron radiation.

100 picosecond X-ray snapshots visualizing the structural dynamics of macromolecular systems are now routinely available at synchrotron sources. A wealth of fundamental processes in photochemistry, condensed matter physics and biology, however, occur on considerably faster time scales. Standard experimental protocols at synchrotron sources cannot provide structural information with faster temporal resolution as these are limited by the duration of the electron bunch within the synchrotron ring. By walking the timing of femtosecond laser photolysis through a (much longer) X-ray pulse in steps of a few picoseconds, structural information on ultrafast dynamics may be retrieved from a set of X-ray scattering images, initially through deconvolution and subsequently through refinement. This experimental protocol promises immediate improvements in the temporal resolution available at synchrotron sources, facilitating the study of a number of rapid complex photochemical processes. Combined with techniques which reshape the X-ray probe pulse, the accessible temporal domain could further be extended to near-picosecond resolution.

High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle.

Bacteriorhodopsin is the simplest known photon-driven proton pump and as such provides a model for the study of a basic function in bioenergetics. Its seven transmembrane helices encompass a proton translocation pathway containing the chromophore, a retinal molecule covalently bound to lysine 216 through a protonated Schiff base, and a series of proton donors and acceptors. Photoisomerization of the all-trans retinal to the 13-cis configuration initiates the vectorial translocation of a proton from the Schiff base, the primary proton donor, to the extracellular side, followed by reprotonation of the Schiff base from the cytoplasm. Here we describe the high-resolution X-ray structure of an early intermediate in the photocycle of bacteriorhodopsin, which is formed directly after photoexcitation. A key water molecule is dislocated, allowing the primary proton acceptor, Asp 85, to move. Movement of the main-chain Lys 216 locally disrupts the hydrogen-bonding network of helix G, facilitating structural changes later in the photocycle.

Femtosecond time resolution in x-ray diffraction experiments.

This paper presents the theoretical background for a synthesis of femtosecond spectroscopy and x-ray diffraction. When a diffraction quality crystal with 0.1-0.3 mm overall dimensions is photoactivated by a femtosecond laser pulse (physical length = 0.3 microm), the evolution of molecules at separated points in the crystal will not be simultaneous because a finite time is required for the laser pulse to propagate through the body of the crystal. Utilizing this lack of global crystal synchronization, topographic x-ray diffraction may enable femtosecond temporal resolution to be achieved from reflection profiles in the diffraction pattern with x-ray exposures of picosecond or longer duration. Such x-ray pulses are currently available, and could be used to study femtosecond reaction dynamics at atomic resolution on crystals of both small- and macromolecules. A general treatment of excitation and diffraction geometries in relation to spatial and temporal resolution is presented.