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Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds., Trypanosoma brucei inosine-5′-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 Å room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

Nature Communications 10(1), 11 (2019). doi:10.1038/s41467-019-12955-3, The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs., Published by Nature Publishing Group UK, [London]

The journal of physical chemistry letters 10(3), 441 - 446 (2019). doi:10.1021/acs.jpclett.8b03595, X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn$^{2+}$ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron cascade effects., Published by ACS, Washington, DC

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Background Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. Conclusions MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data. Electronic supplementary material The online version of this article (10.1186/s12915-018-0524-5) contains supplementary material, which is available to authorized users.

A new approach of using lipidic cubic phase as a carrier matrix for delivering soluble protein microcrystals for serial crystallography helps to dramatically reduce protein consumption. The structures of two soluble test proteins have been determined by this method using less than 0.1 mg of each protein., Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

Viscous sample delivery that decreases the net protein consumed in serial femtosecond crystallography is described. The agarose stream has a low background, is compatible with membrane proteins and can be used at a wide range of temperatures., Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

Scientific Data 4:170048 doi: 10.1038/sdata201748 (2017); Published 11 April 2017; Updated 24 October 2017. The Data Descriptor incorrectly states the number of normal incidences used to generate the plot in Fig. 4b as 209. This plot was generated from 32 normal incidence cases.

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis ‘in-action’ by ‘mix-and-inject serial crystallography’. Specifically, we follow the catalytic reaction of theMycobacterium tuberculosisα-lactamase with the 3rdgeneration antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.SynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

Proceedings of the National Academy of Sciences of the United States of America 114(9), 2247 – 2252 (2017). doi:10.1073/pnas.1609243114, To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm$^3$ in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm$^3$ in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach., Published by National Acad. of Sciences, Washington, DC

Biophysical journal 112(3), 506a - 507a (2017). doi:10.1016/j.bpj.2016.11.2740, Published by Cell Press, Cambridge, Mass.

Femtosecond X-ray crystallography allows structural analysis of a difficult-to-crystallize fusion protein that is a potential component of a candidate HIV-1 subunit vaccine., CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions. peerReviewed

Nature 530(7589), 202 - 206(2016). doi:10.1038/nature16949, The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 $\mathring{A}$ limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 $\mathring{A}$ as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 $\mathring{A}$. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing., Published by Nature Publ. Group, London [u.a.]

Many biological processes depend on detecting and responding to light. The response is often mediated by a structural change in a protein that begins when absorption of a photon causes isomerization of a chromophore bound to the protein. Pande et al. used x-ray pulses emitted by a free electron laser source to conduct time-resolved serial femtosecond crystallography in the time range of 100 fs to 3 ms. This allowed for the real-time tracking of the trans-cis isomerization of the chromophore in photoactive yellow protein and the associated structural changes in the protein.Science, this issue p. 725A 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.

Background: Rubisco activase has been linked to the inhibition of net photosynthesis upon warming. Results: The structure of the C-terminal domain adopts an unusually elongated shape. Conclusions: Reactivation of Rubisco may involve movement of a paddle-like extension. Significance: This work will aid in gaining a better understanding of Rubisco regulation., The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO2 fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question.

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution., Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.

Nature structural & molecular biology 22(3), 265 - 268(2015). doi:10.1038/nsmb.2965, Bifunctional μ- and ​δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human ​δ-OR bound to the bifunctional ​δ-OR antagonist and ​μ-OR agonist tetrapeptide ​H-Dmt-Tic-Phe-Phe-NH2 (​DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between ​H-Dmt and ​Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics., Published by Nature Publishing Group, London [u.a.]

Diffraction Before Destruction A bottleneck in x-ray crystallography is the growth of well-ordered crystals large enough to obtain high-resolution diffraction data within an exposure that limits radiation damage. Serial femtosecond crystallography promises to overcome these constraints by using short intense pulses that out-run radiation damage. A stream of crystals is flowed across the free-electron beam and for each pulse, diffraction data is recorded from a single crystal before it is destroyed. Redecke et al. (p. 227 , published online 29 November; see the Perspective by Helliwell ) used this technique to determine the structure of an enzyme from Trypanosoma brucei , the parasite that causes sleeping sickness, from micron-sized crystals grown within insect cells. The structure shows how this enzyme, which is involved in degradation of host proteins, is natively inhibited prior to activation, which could help in the development of parasite-specific inhibitors.

X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.

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., Serial femtosecond crystallography is an X-ray free-electron-laser-based method that uses X-ray bursts to determine protein structures. Here the authors present the structure of a photosynthetic reaction centre, an integral membrane protein, achieved with no sign of X-ray-induced radiation damage.

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.

Size Matters Less X-ray crystallography is a central research tool for uncovering the structures of proteins and other macromolecules. However, its applicability typically requires growth of large crystals, in part because a sufficient number of molecules must be present in the lattice for the sample to withstand x-ray—induced damage. Boutet et al. (p. 362 , published online 31 May) now demonstrate that the intense x-ray pulses emitted by a free-electron laser source can yield data in few enough exposures to uncover the high-resolution structure of microcrystals.

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded(1-3). It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source(4). We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes(5). More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (similar to 200 nm to 2 mm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes(6). This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single−shot X−ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle−size distribution. The data were collected at the Linac Coherent Light Source (the first hard−X−ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single `still' diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal−size effects. These results were compared with single−crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation−damage reduction and for time−resolved diffraction studies at room temperature are discussed

A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F(1)-ATPase gamma-subunit rotation was determined to be 7.62 +/- 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg(2+)-ATPase k(cat) of 130 s(-)(1) (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F(1) was 63.3 +/- 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F(1), resulting in direct implications for its rotational mechanism.

The mechanism of atrazine binding and its modification by Chelex-100-induced Ca2+ depletion and proteolytic degradation by trypsin, was analyzed in PS II membrane fragments from spinach. It was found: 1) Chelex-100 treatment leads in a comparatively slow process (t1/2 = 5 - 10 min) to Ca2+ re­ moval from a site that is characterized by a high affinity as reflected by KD values of the order of 10-7 M. The number of these binding sites was found to be almost one per PS II in samples washed twice with Ca2+ -free buffer. 2) Chelex-100 treatment does not affect the affinity of atrazine binding but increases the susceptibility to proteolytic attack by trypsin. 3) The electron transport activity is only slightly affected by Chelex-100 treatment. 4) The atrazine binding exhibits a rather small T-dependence within the physiological range of 7 °C to 27 °C. The implications of these findings for herbicide binding are discussed.

The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10-15 c-subunits is commonly thought to drive rotation of the rotor moiety (c(10-14)gammaepsilon) relative to stator moiety (alpha(3)beta(3)deltaab(2)). Here we report the isolation and crystallization of the c(14)-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 A. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase.

Comparative studies in thylakoids and oxygen-evolving Triton X-100 PS II membrane fragments reveal: (i) There exists one high-affinity atrazine binding site per PS II in both preparations. The affinity is reduced in PS II membrane fragments, (ii) The susceptibility to tryptic attack on atrazine binding and p-BQ-mediated electron transport is markedly reduced by CaCl2 in PS II membrane fragments, but no protection is observed in thylakoids. NH2OH and K3[Fe(CN)6] both reduce the binding affinity in PS II membrane fragments. The action of NH2OH is invariant to CaCl2 addition. The implications of these findings for functional studies with PS II membrane fragments are discussed.

The integral membrane protein complex, menaquinol:fumarate oxidoreductase (mQFR) has been purified, identified and characterized from the thermophilic green filamentous anoxygenic photosynthetic bacterium Chloroflexus aurantiacus. The complex is composed of three subunits: a 74 kDa flavoprotein that contains a covalently bound flavin adenine dinucleotide, a 28 kDa iron-sulfur cluster-containing polypeptide, and a 27 kDa transmembrane polypeptide, which is also the binding site of two b-type hemes and two menaquinones. The purified complex has an apparent molecular mass of 260 kDa by blue-native PAGE, which is indicative of a native homodimeric form. The isolated complex is active in vitro in both fumarate reduction and succinate oxidation. It has been analyzed by visible absorption, redox titration, chemical analysis and EPR spectroscopy. In addition, phylogenetic analysis shows that the QFR of both C. aurantiacus and Chlorobium tepidum are most closely related to those found in the delta-proteobacteria. The purified enzyme was crystallized and X-ray diffraction data obtained up to 3.2 Å resolution.

The need for a renewable and sustainable energy source has become apparent, and focusing on a bioinspired approach emphasizing the water splitting mechanism in photosynthesis is a challenging, yet practical and attainable strategy. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a biomimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). In nature, one of Photosystem II's core proteins, D1, is degraded every half hour in the presence of sunlight. D1's sensitivity photodamage resides in triplet state formation of chlorophyll P680+, the primary donor of PSII. Our project aims to build the heart of the OEC, including the Mn4CaCl metal cluster and its protein environment in stable DNA nanocages, which can be connected to a photostable artificial reaction center that performs light-induced charge separation. The peptide sequences responsible for coordinating the cluster have been identified through x-ray structure analysis. RTruncated regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphine chloride, have been implemented for testing of the encapsulation of a metal-containing molecule inside of the DNA cage. Upon reduction of the metal through coordination of two orthogonally oriented peptides covalently attached to the DNA tetrahedron which contain terminal histidine residues, iron (III) becomes EPR active and the assembly can be analyzed electrochemically. Upon assembly of the complete metal-containing center (both porphyrin molecule and aOEC), x-ray crystallography, EPR, and electrochemistry will be used to test functionality in the stable DNA framework.

Membrane proteins are of extreme relevance for all living cells. They are important for energy supply of the cells, transport across membranes, cell communication and play also vital roles in the pathogenesis of viruses and bacteria. Despite their important function, less than 300 membrane protein structures have been determined to date. One of the major limiting steps for the structure determination is the growth of well-ordered single crystals. New results of a rationale design of membrane protein crystallization are presented, that include screening for the reversibility of crystallization conditions, determination of phase diagrams, and determination of early events in nucleation and crystal growth by a combination of dynamic light scattering, SONICC, and tryptophan fluorescence microscopy. Nanocrystals detected, can either be used as seeds for growth of large single crystals or used for structure determination by femtosecond nanocrystallography. The latter method is based on a novel concept for structure determination, where X-ray diffraction “snapshots” are collected from a fully hydrated stream of nanocrystals, using femtosecond pulses from the world's first high energy X-ray free-electron laser, the Linac Coherent Light Source. The experiments show the proof of concept that diffraction of nanocrystals that contain only 100-10 000 membrane proteins can be observed and used for structure determination of large membrane protein complexes [1]. The femtosecond pulses are 1012 stronger than 3rd generation synchrotron sources and destroy any material that is placed in its focus, but as the femtosecond pulses are briefer than the time-scale of most damage processes, the method overcomes the problem of damage in crystallography. Femtosecond crystallography also opens new avenues for determination of molecular movies of protein dynamics.Reference: [1] Chapman et al, 2011 Nature, 470, 73-77.