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Recently, significant technological innovations have enabled the measurement of both X-ray and electron diffraction from protein microcrystals. These new microcrystallography experiments are useful when large crystals cannot be obtained, but also in other cases, such as when large crystals suffer from long-range disorder, or when uniform perturbations need to be applied rapidly to the entire crystal volume. Optimizing the preparation of protein microcrystals for this new class of experiments presents new challenges for crystallographers, who have traditionally sought to grow large, single crystals. To better understand these new challenges, we optimized the production of microcrystalline samples of cyclophilin A (CypA), starting from conditions that produced millimeter scale crystals. Next, we used these microcrystals to determine CypA structures by serial femtosecond crystallography (SFX) at two XFEL lightsources, and by microcrystal electron diffraction (microED) in an electron cryomicroscope. Here, I will present our optimization strategy for protein microcrystallization, and compare the results of X-ray and electron microcrystallography experiments with CypA. I will focus on the unique caveats of sample delivery for each method, and compare the resulting structures. The goal will be to provide insight into which microcrystallography experiment is most appropriate for which types of samples, and to share our experience with sample preparation and delivery for each type of experiment.

Achieving structurally well-defined catalytic species requires a fundamental understanding of surface chemistry. Detailed structural characterization of the catalyst binding sites in situ, such as single site catalysts on silica supports, is technically challenging or even unattainable. Octadecyltrioxysilane (OTOS) monolayers formed from octadecyltrimethoxysilane (OTMS) at the air-liquid interface after hydrolysis and condensation at low pH were chosen as a model system of surface binding sites in silica-supported Zn(2+) catalysts. We characterize the system by grazing incidence X-ray diffraction, X-ray reflectivity (XR), and X-ray fluorescence spectroscopy (XFS). Previous X-ray and infrared surface studies of OTMS/OTOS films at the air-liquid interface proposed the formation of polymer OTOS structures. According to our analysis, polymer formation is inconsistent with the X-ray observations and structural properties of siloxanes; it is energetically unfavorable and thus highly unlikely. We suggest an alternative mechanism of hydrolysis/condensation in OTMS leading to the formation of structurally allowed cyclic trimers with the six-membered siloxane rings, which explain well both the X-ray and infrared results. XR and XFS consistently demonstrate that tetrahedral [Zn(NH3)4](2+) ions bind to hydroxyl groups of the film at a stoichiometric ratio of OTOS:Zn ∼ 2:1. The high binding affinity of zinc ions to OTOS trimers suggests that the six-membered siloxane rings are binding locations for single site Zn/SiO2 catalysts. Our results show that OTOS monolayers may serve as a platform for studying silica surface chemistry or hydroxyl-mediated reactions.

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase versus warfarin, and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum (BDF) and difenacoum (DiF) were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raises the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we will summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

Gram-negative bacteria possess a double membrane system, with the external leaflet of the outer membrane composed of lipopolysaccharides (LPS) that act as the first barrier to drugs. The importance of studying the permeability of LPS barrier comes from its correlation with strain susceptibility. In this study, we extracted LPS from two isogenic Salmonella sp strains, that contains alterations in the lipid A portion regulated by the phoPQ-system. Interaction between phoP- (LPS with no modification) or phoPc (palmitoylation of the 3-hydroxyl group in the 3-OH-myristyl residue, addition of 4-aminoarabinose to the 4' phosphate group and addition of a 2-hydroxy group to the myristate residue at the position 3') LPS and novobiocin was evaluated by using Langmuir monolayers. Novobiocin was chosen due to its size (612 Da), large enough to permeate cells throughout the LPS, and not through the porin channels. MIC of novobiocin for phoP- was 14 μg mL-1 and for phoPc, 40 μg mL-1. p-A isotherms indicated that phoP- occupies a limiting area of 150A2, while for phoPc this value is 175A2. However, addition of Mg2+ promotes condensation of 20A2 for phoP-, and 70A2 for phoPc. Epifluorescence microscopy reveals phoP- has fluid-like morphology .In contrast, phoPc displays circular-shaped domains that increase in size and number with the compression. The addition of novobiocin to the subphase promoted slight changes in phoP- morphology. However, for phoPc monolayer, changes in morphology were observed for higher concentrations of novobiocin at which domains started to aggregate generating structures like a "pearl necklace". Structural organization was evaluated by GIXD, and data corroborates the one obtained by epifluorescence, in which novobiocin interaction promotes liquid-crystalline phase formation. Electron density profile probed that novobiocin interacts with phoP- hydrophobic portion, but much higher concentration is needed to permeate phoPc.

A novel supported bilayer system at the air-water interface has been developed for use in synchrotron X-ray scattering experiments in order to drastically increase signal to noise ratio. This system gives a significant resolution improvement comparing to more traditional solid-supported bilayer system and is better suited for specular X-Ray reflectivity and grazing incidence X-ray off-specular scattering. Using this system we have successfully characterized anionic bilayers mimetic of Gram-positive bacterial membranes interacting with engineered biomimetic oligomers (peptoids), zwitterionic bilayers interacting with human islet amyloid polypeptide (IAPP), and insertion differences between zwitterionic membranes with and without cholesterol and the glycoprotein-41 subunit fragment of the viral envelope glycoprotein-160. Results from anionic and net neutral bilayers correspond to previous studies on monolayer insertion studies, where peptoid ML 2-6 acts by associating throughout the lipophilic regions and zwitterionic bilayers without cholesterol were completely penetrated by the GP-41 fusion peptide. Resolution of the dynamic formation of a zwitterionic lipid bilayer system in minute scale time resolution via Grazing Incidence X-Ray Off-Specular Scattering (GIXOS) was also observed, and found to be in agreement with the proposed mechanisms associated with vesicle fusion on a solid support.

© 2015 Biophysical Society. Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.

Pulmonary surfactant (PS) is the complex mixture of lipids and proteins that forms a thin film on the liquid layer that lines the alveolar air-sacks of the lungs. When compressed by the decreasing alveolar surface area during exhalation, the surfactant films reduce surface tension to exceptionally low levels. This behavior in situ contrasts with the performance of spread monolayers in vitro that contain the complete set of surfactant constituents, which collapse promptly when compressed below the equilibrium spreading pressure. The structural characteristics that provide the basis for this functional difference remain controversial. The studies here used X-ray reflectivity to compare the structure of adsorbed films and spread monolayers of extracted calf surfactant on the air-water interface. Our results show the presence in the adsorbed film of additional double layers of distinct electron densities underneath the interfacial monolayer. These results support previous evidence that adsorbed films of pulmonary surfactant have multilamellar thickness, and that the additional layers may have functional significance.