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The drug development landscape is expanding to include drug modalities such as PROteolysis-TArgeting Chimeras (PROTACs) and peptides, offering possibilities for previously intractable biological targets. However, with their size and chemical nature, they diverge from established frameworks for the prediction of oral bioavailability. This evolution to larger and more complex molecules necessitates new methodologies and prediction models to continuously expand on bioavailability guidelines. We describe the high-capacity adoption of two chromatographic physicochemical assays and their application for iterative compound optimization to achieve oral bioavailability. We further describe how these data underpin the continuous refinement of internal machine learning models, which guide compound synthesis decisions in the molecular design phase. Based on data for a set of 691 PROTACs, and two project examples, we confirm a sweet spot for oral bioavailability at log D values higher than the norm for small molecules and show how experimental data and prediction models synergize to effectively drive chemistry optimization.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography 1 using an X-ray free-electron laser 2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Virtual screening is a standard tool in Computer-Assisted Drug Design (CADD). Early in a project, it is typical to use ligand-based similarity search methods to find suitable hit molecules. However, the number of compounds which can be screened and the time required are usually limited by computational resources. We describe here a high-throughput virtual screening project using 3D similarity (FastROCS) and automated evaluation workflows on Orion, a cloud computing platform. Cloud resources make this approach fully scalable and flexible, allowing the generation and search of billions of virtual molecules, and give access to an explicit 3D virtual chemistry space not available before. We discuss the impact of the size of the search space with respect to finding novel chemical hits and the size of the required hit list, as well as computational and economical aspects of resource scaling.

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which X-ray solution scattering data are collected from particles in solution using ultrashort X-ray exposures generated by a free-electron laser (FEL). FXS experiments overcome the low data-to-parameter ratios associated with traditional solution scattering measurements by providing several orders of magnitude more information in the final processed data. Here we demonstrate the practical feasibility of FEL-based FXS on a biological multiple-particle system and describe data-processing techniques required to extract robust FXS data and significantly reduce the required number of snapshots needed by introducing an iterative noise-filtering technique. We showcase a successful ab initio electron density reconstruction from such an experiment, studying the Paramecium bursaria Chlorella virus (PBCV-1)., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which solution scattering data are collected using X-ray exposures below rotational diffusion times, resulting in angularly anisotropic X-ray snapshots that provide several orders of magnitude more information than traditional solution scattering data. Such experiments can be performed using the ultrashort X-ray pulses provided by a free-electron laser source, allowing one to collect a large number of diffraction patterns in a relatively short time. Here, we describe a test data set for FXS, obtained at the Linac Coherent Light Source, consisting of close to 100 000 multi-particle diffraction patterns originating from approximately 50 to 200 Paramecium Bursaria Chlorella virus particles per snapshot. In addition to the raw data, a selection of high-quality pre-processed diffraction patterns and a reference SAXS profile are provided.

The photochemistry of halomethanes is fascinating for the complex cascade reactions toward either the parent or newly synthesized molecules. Here, we address the structural rearrangement of photodissociated CH 2 IBr in methanol and cyclohexane, probed by time-resolved X-ray scattering in liquid solution. Upon selective laser cleavage of the C-I bond, we follow the reaction cascade of the two geminate geometrical isomers, CH 2 I-Br and CH 2 Br-I. Both meta-stable isomers decay on different time scales, mediated by solvent interaction, toward the original parent molecule. We observe the internal rearrangement of CH 2 Br-I to CH 2 I-Br in cyclohexane by extending the time window up to 3 μs. We track the photoproduct kinetics of CH 2 Br-I in methanol solution where only one isomer is observed. The effect of the polarity of solvent on the geminate recombination pathways is discussed.

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 (RC vir ). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RC vir 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

X-ray scattering images collected on timescales shorter than rotation diffusion times using a (partially) coherent beam result in a significant increase in information content in the scattered data. These measurements, named fluctuation X-ray scattering (FXS), are typically performed on an X-ray free-electron laser (XFEL) and can provide fundamental insights into the structure of biological molecules, engineered nanoparticles or energy-related mesoscopic materials beyond what can be obtained with standard X-ray scattering techniques. In order to understand, use and validate experimental FXS data, the availability of basic data characteristics and operational properties is essential, but has been absent up to this point. In this communication, an intuitive view of the nature of FXS data and their properties is provided, the effect of FXS data on the derived structural models is highlighted, and generalizations of the Guinier and Porod laws that can ultimately be used to plan experiments and assess the quality of experimental data are presented.

Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested., (Copyright © 2015, American Association for the Advancement of Science.)

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.

Helium nanodroplets are considered ideal model systems to explore quantum hydrodynamics in self-contained, isolated superfluids. However, exploring the dynamic properties of individual droplets is experimentally challenging. In this work, we used single-shot femtosecond x-ray coherent diffractive imaging to investigate the rotation of single, isolated superfluid helium-4 droplets containing ~10(8) to 10(11) atoms. The formation of quantum vortex lattices inside the droplets is confirmed by observing characteristic Bragg patterns from xenon clusters trapped in the vortex cores. The vortex densities are up to five orders of magnitude larger than those observed in bulk liquid helium. The droplets exhibit large centrifugal deformations but retain axially symmetric shapes at angular velocities well beyond the stability range of viscous classical droplets., (Copyright © 2014, American Association for the Advancement of Science.)

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.

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.

X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis 1 . For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information 1-4 . Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology 5 should enable structural determination from submicrometre protein crystals with atomic resolution.

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein's resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein's conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein's reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and time-resolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon.

Membrane proteins are embedded in a lipid bilayer and maintain strong interactions with lipid molecules. Tightly bound lipids are responsible for vertical positioning and integration of proteins in the membrane and for assembly of multisubunit complexes and occasionally act as substrates. In this work we present the lipidic sponge phase crystal structure of the reaction center from Blastochloris viridis to 1.86 A, which reveals lipid molecules interacting with the protein surface. A diacylglycerol molecule is bound, through a thioether bond, to the N-terminus of the tetraheme cytochrome c subunit. From the electron density recovered at the Q(B) site and the observed change in recombination kinetics in lipidic sponge phase-grown crystals, the mobile ubiquinone appears to be displaced by a monoolein molecule. A 36 A long electron density feature is observed at the interface of transmembrane helices belonging to the H- and M-subunits, probably arising from an unidentified lipid.

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.

Ultrafast time-resolved wide angle x-ray scattering from chemical reactions in solution has recently emerged as a powerful technique for determining the structural dynamics of transient photochemical species. Here we examine the structural evolution of photoexcited CH(2)I(2) in the nonpolar solvent cyclohexane and draw comparisons with a similar study in the polar solvent methanol. As with earlier spectroscopic studies, our data confirm a common initial reaction pathway in both solvents. After photoexcitation, CH(2)I(2) dissociates to form CH(2)I* + I*. Iodine radicals remaining within the solvent cage recombine with a nascent CH(2)I* radical to form the transient isomer CH(2)I-I, whereas those which escape the solvent cage ultimately combine to form I(2) in cyclohexane. Moreover, the transient isomer has a lifetime approximately 30 times longer in the nonpolar solvent. Of greater chemical significance is the property of time-resolved wide angle x-ray diffraction to accurately determine the structure of the of CH(2)I-I reaction intermediate. Thus we observe that the transient iodine-iodine bond is 0.07 A+/-0.04 A shorter in cyclohexane than in methanol. A longer iodine-iodine bond length for the intermediate arises in methanol due to favorable H-bond interaction with the polar solvent. These findings establish that time-resolved x-ray diffraction has sufficient sensitivity to enable solvent dependent structural perturbations of transient chemical species to be accurately resolved.

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.