Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results. Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY  II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input. Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Biophysical investigation into the antibacterial action of modelin-5-NH2.

Modelin-5-CONH2 (M5-NH2) is a synthetic antimicrobial peptide, which was found to show potent activity against Bacillus subtilis (minimum lethal concentration = 8.47 µM) and to bind strongly to membranes of the organism (Kd = 10.44 µM). The peptide adopted high levels of amphiphilic α-helical structure in the presence of these membranes (>50%), which led to high levels of insertion (Δπ ≥ 8.0 mN m-1). M5-NH2 showed high affinity for anionic lipid (Kd = 7.46 µM) and zwitterionic lipid (Kd = 14.7 µM), which drove insertion into membranes formed from these lipids (Δπ = 11.5 and 3.5 mN m-1, respectively). Neutron diffraction studies showed that M5-NH2 inserted into B. subtilis membranes with its N-terminal residue, L16, located 5.5 Å from the membrane centre, in the acyl chain region of these membranes, and promoted a reduction in membrane thickness of circa 1.8 Å or 5% of membrane width. Insertion into B. subtilis membranes by the peptide also promoted other effects associated with membrane thinning, including increases in membrane surface area (Cs-1 decreases) and fluidity (ΔGmix > 0 to ΔGmix < 0). Membrane insertion and thinning by M5-NH2 induced high levels of lysis (>55%), and it is speculated that the antibacterial action of the peptide may involve the toroidal pore, carpet or tilted-type mechanism of membrane permeabilization.

Lipid bilayer position and orientation of novel carprofens, modulators of γ-secretase in Alzheimer's disease.

γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-ß peptides. Amyloid-ß peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.

Impact of the ceramide subspecies on the nanostructure of stratum corneum lipids using neutron scattering and molecular dynamics simulations. Part I: impact of CER[NS].

For this study mixtures based on the ceramides [NS] (NS = non-hydroxy-sphingosine) and [AP] (AP = α-hydroxy-phytosphingosine) in a 2:1 and 1:2 ratio, together with cholesterol and lignoceric acid, were investigated. These mixtures are modelling the uppermost skin layer, the stratum corneum. Neutron diffraction, utilizing specifically deuterated ceramide molecules, was used to obtain a maximum amount of experimental detail. Highly detailed molecular dynamics simulations were used to generate even more information from the experimental data. It was possible to observe a single lamellar phase for both systems. They had a lamellar repeat distance of 5.43 ±â€¯0.05 nm for the [NS]/[AP] 2:1 and a slightly shorter one of 5.34 ±â€¯0.05 nm for the 1:2 system. The structure and water content was uninfluenced by excess humidity. Both the experimental and simulation data indicated slightly tilted ceramides, with their C24 chains overlapping in the lamellar mid-plane. This arrangement is well comparable to systems investigated before. The structure of both systems, except for the differing repeat distance, looks similar at first. However, on a smaller scale there were various distinct differences, demonstrating only low redundancy between the different ceramide species, despite only minor chemical differences. The mainly ceramide [AP] determined 1:2 system has a slightly smaller repeat distance. This is a result of a tighter arrangement of the lipids chain along the bilayer normal and increased overlapping of the long chains in the lamellar middle. For the CER[NS] some novel features could be shown, despite it being the overall most investigated ceramide. These include the low adaptability to changed lateral interactions, leading to an increased chain opening. This effect could explain its low miscibility with other lipids. The investigated model systems allows it to directly compare results from the literature which have used ceramide [NS] to the most recent studies using the phytosphingosine ceramides such as ceramide [AP].

Investigation of a CER[NP]- and [AP]-Based Stratum Corneum Modeling Membrane System: Using Specifically Deuterated CER Together with a Neutron Diffraction Approach.

Neutron diffraction was used as a tool to investigate the lamellar as well as molecular nanostructure of ceramide-[NP]/ceramide-[AP]/cholesterol/lignoceric acid model systems with a nativelike 2:1 ratio and a 1:2 ratio to study the influence of the ceramide-[AP]. By using mixtures together with cholesterol and free fatty acids as well as a humidity and temperature chamber while measuring, natural conditions were simulated as closely as possible. Despite its simplicity, the system simulated the native stratum corneum lipid matrix fairly closely, showing a similar lamellar thickness with a repeat distance of 5.45 ± 0.1 nm and a similar arrangement with overlapping long C24 chains. Furthermore, despite the very minor chemical difference between ceramide-[NP] and ceramide-[AP], which is only a single OH group, it was possible to demonstrate substantial differences between the structural influence of the two ceramides. Ceramide-[AP] could be concluded to be arranged in such a way that its C24 chain in both ratios is somehow shorter than that of ceramide-[NP], not overlapping as much with the opposite lamellar leaflet. Furthermore, in the unnatural 1:2 ratio, the higher ceramide-[AP] content causes an increased tilt of the ceramide acyl chains. This leads to even less overlapping within the lamellar midplane, whereas the repeat distance stays the same as for the ceramide-[NP]-rich system. In this nativelike 2:1 ratio, the chains are arranged mostly straight, and the long C24 chains show a broad overlapping region in the lamellar midplane.

Alcohol Interactions with Lipid Bilayers.

We investigate the structural changes to lipid membrane that ensue from the addition of aliphatic alcohols with various alkyl tail lengths. Small angle neutron diffraction from flat lipid bilayers that are hydrated through water vapor has been employed to eliminate possible artefacts of the membrane curvature and the alcohol's membrane-water partitioning. We have observed clear changes to membrane structure in both transversal and lateral directions. Most importantly, our results suggest the alteration of the membrane-water interface. The water encroachment has shifted in the way that alcohol loaded bilayers absorbed more water molecules when compared to the neat lipid bilayers. The experimental results have been corroborated by molecular dynamics simulations to reveal further details. Namely, the order parameter profiles have been fruitful in correlating the mechanical model of structural changes to the effect of anesthesia.

Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration.

Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix.

Influence of a Novel Dimeric Ceramide Molecule on the Nanostructure and Thermotropic Phase Behavior of a Stratum Corneum Model Mixture.

The stratum corneum (SC) is the outermost layer of the skin and is composed of a multilayered assembly of mostly ceramids (Cer), free fatty acids, cholesterol (Chol), and cholesterol sulfate (Chol-S). Because of the tight packing of these lipids, the SC features unique barrier properties defending the skin from environmental influences. Under pathological conditions, where the skin barrier function is compromised, topical application of molecules that rigidify the SC may lead to a restored barrier function. To this end, molecules are required that incorporate into the SC and bring back the original rigidity of the skin barrier. Here, we investigated the influence of a novel dimeric ceramide (dim-Cer) molecule designed to feature a long, rigid hydrocarbon chain ideally suited to forming an orthorhombic lipid phase. The influence of this molecules on the thermotropic phase behavior of a SC mixture consisting of Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %) was studied using a combination of neutron diffraction and 2H NMR spectroscopy. These methods provide detailed insights into the packing properties of the lipids in the SC model mixture. Dim-Cer remains in an all-trans state of the membrane-spanning lipid chain at all investigated temperatures, but the influence on the phase behavior of the other lipids in the mixture is marginal. Biophysical experiments are complemented by permeability measurements in model membranes and human skin. The latter, however, indicates that dim-Cer only partially provides the desired effect on membrane permeability, necessitating further optimization of its structure for medical applications.

Synthesis of specific deuterated derivatives of the long chained stratum corneum lipids [EOS] and [EOP] and characterization using neutron scattering.

The synthesis of specific deuterated derivatives of the long chained ceramides [EOS] and [EOP] is described. The structural differences with respect to the natural compounds are founded in the substitution of the 2 double bonds containing linoleic acid by a palmitic acid branched with a methyl group in 10-position. The specific deuteration is introduced both in the branched and in the terminal methyl group, which was realized by common methods of successive deuteration of carboxylic groups in 3 steps. These modified fatty acids resp. the corresponding ceramides [EOS] and [EOP] were prepared for neutron scattering investigations. First results of these investigations were presented in this manuscript showing that the deuterated compounds could be detected in the stratum corneum lipid model membranes. The deuterated ceramides [EOS] and [EOP] are valuable tools to investigate the influence of these long chained ceramide species on the nanostructure of stratum corneum lipid model membranes.

Phase separation in ceramide[NP] containing lipid model membranes: neutron diffraction and solid-state NMR.

The stratum corneum is the outermost layer of the skin and protects the organism against external influences as well as water loss. It consists of corneocytes embedded in a mixture of ceramides, fatty acids, and cholesterol in a molar ratio of roughly 1 : 1 : 1. The unique structural and compositional arrangement of these stratum corneum lipids is responsible for the skin barrier properties. Many studies investigated the organization of these barrier lipids and, in particular, the exact conformation of ceramides. However, so far no consensus has been reached. In this study, we investigate a model system comprised of N-(non-hydroxy-tetracosanoyl)-phytosphingosine/cholesterol/tetracosanoic acid (CER[NP]-C24/CHOL/TA) at a 1 : 1 : 1 molar ratio using neutron diffraction and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Deuterated variants of all three lipid components of the model system were used to enable their separate investigation in the NMR spectra and quantification of the amount of molecules in each phase. Neutron scattering experiments show the coexistence of two lipid phases at low temperatures with repeat spacings of 54.2 Å and 43.0 Å at a physiological skin temperature of 32 °C. They appear to be indistinguishable in the 2H NMR spectra as both phases are crystalline and ceramide molecules do not rotate around their long axis on a microsecond timescale. The evolution of these phases upon heating is followed and with increasing temperature fluid and even isotropically mobile molecules are observed. A model of the organization of the lamellar phases is proposed in which the thicker phase consists of CER[NP]-C24 in a hairpin conformation mixed with CHOL and TA, while the phase with a repeat spacing of 43.0 Å contains CER[NP]-C24 in a V-shape conformation.

Synthesis of specifically deuterated ceramide [AP]-C18 and its biophysical characterization using neutron diffraction.

The very heterogeneous group of ceramides is known to be mandatory for proper barrier functions of the outermost layer of mammalian skin, referred to as stratum corneum (SC). The synthesis of a specifically deuterated ceramide [AP]-C18 variant is described. The synthesized ceramide contains the racemic forms of the α hydroxy fatty acid. For the biophysical implementation, the received diastereomeric ceramide was applied in a neutron diffraction experiment. Therefore, a SC lipid model membrane was prepared containing the described ceramide (CER), cholesterol (CHOL), stearic acid (SA), and cholesterol sulfate (ChS) in a ratio of 55/25/15/5wt%. Thus, we were able to localize the deuterated molecule part within the bilayers. In the process, a short-periodicity phase (SPP) was observed with a unit cell scale of about 44Å. For the first time, we were able to confirm former ideas concerning the arrangement of the CER within this quaternary lipid model membrane.

Influence of the penetration enhancer isopropyl myristate on stratum corneum lipid model membranes revealed by neutron diffraction and 2H NMR experiments.

The stratum corneum (SC) provides the main barrier properties in native skin. The barrier function is attributed to the intercellular lipids, forming continuous multilamellar membranes. In this study, SC lipid membranes in model ratios were enriched with deuterated lipids in order to investigate structural and dynamical properties by neutron diffraction and 2H solid-state NMR spectroscopy. Further, the effect of the penetration enhancer isopropyl myristate (IPM) on the structure of a well-known SC lipid model membrane containing synthetically derived methyl-branched ceramide [EOS], ceramide [AP], behenic acid and cholesterol (23/10/33/33wt%) was investigated. IPM supported the formation of a single short-periodicity phase (SPP), in which we determined the molecular organization of CER[AP] and CER[EOS]-br for the first time. Furthermore, the thermotropic phase behavior of the lipid system was analyzed by additional neutron diffraction studies as well as by 2H solid-state NMR spectroscopy, covering temperatures of 32°C (physiological skin temperature), 50°C, and 70°C with a subsequent cooldown back to skin temperature. Both techniques revealed a phase transition and a hysteresis effect. During the cooldown, Bragg peaks corresponding to a long-periodicity phase (LPP) appeared. Additionally, 2H NMR revealed that the IPM molecules are isotopic mobile at all temperatures.

Localization of methyl-branched ceramide [EOS] species within the long-periodicity phase in stratum corneum lipid model membranes: A neutron diffraction study.

The outermost layer of the mammalian skin, the stratum corneum (SC), is a very thin structure and realizes simultaneously the main barrier properties. The penetration barrier for xenobiotica is mostly represented by a complex lipid matrix. There is great interest in the subject of getting information about the arrangement of the lipids, which are mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). SC lipid model membranes containing synthetically derived lipids in a non-physiological ratio were investigated. To compare the study to a former experiment, a methyl-branched ceramide [EOS] species in presence of the ultra-long chain CER[AP], CHOL and behenic acid (23/10/33/33, wt%) was applied. The membrane structure was studied using the very versatile technique of neutron diffraction. We were able to identify a long-periodicity phase (LPP) with a size of 114Å or 118Å with CER[EOS]-br in a ratio of >60wt% of the ceramides. Furthermore, we figured out two additional coexisting short-periodicity phases (SPP) with repeat distances of 48Å and 45Å, respectively. Partial deuterations of CER[EOS]-br and CER[AP] enabled the localization of the molecules within the multiphase system. CER[EOS]-d3 was present in the LPP, but absent in both SPP. CER[AP]-d3 was determined in both short phases but not localized within the LPP. Besides, we revealed influences of humidity and time with respect to the long-periodicity phase.

Alzheimer's peptide amyloid-ß, fragment 22-40, perturbs lipid dynamics.

The peptide amyloid-ß (Aß) interacts with membranes of cells in the human brain and is associated with Alzheimer's disease (AD). The intercalation of Aß in membranes alters membrane properties, including the structure and lipid dynamics. Any change in the membrane lipid dynamics will affect essential membrane processes, such as energy conversion, signal transduction and amyloid precursor protein (APP) processing, and may result in the observed neurotoxicity associated with the disease. The influence of this peptide on membrane dynamics was studied with quasi-elastic neutron scattering, a technique which allows a wide range of observation times from picoseconds to nanoseconds, over nanometer length scales. The effect of the membrane integral neurotoxic peptide amyloid-ß, residues 22-40, on the in- and out-of-plane lipid dynamics was observed in an oriented DMPC/DMPS bilayer at 15 °C, in its gel phase, and at 30 °C, near the phase transition temperature of the lipids. Near the phase-transition temperature, a 1.5 mol% of peptide causes up to a twofold decrease in the lipid diffusion coefficients. In the gel-phase, this effect is reversed, with amyloid-ß(22-40) increasing the lipid diffusion coefficients. The observed changes in lipid diffusion are relevant to protein-protein interactions, which are strongly influenced by the diffusion of membrane components. The effect of the amyloid-ß peptide fragment on the diffusion of membrane lipids will provide insight into the membrane's role in AD.

The Position of Aß22-40 and Aß1-42 in Anionic Lipid Membranes Containing Cholesterol.

Amyloid-ß peptides interact with cell membranes in the human brain and are associated with neurodegenerative diseases, such as Alzheimer's disease. An emerging explanation of the molecular mechanism, which results in neurodegeneration, places the cause of neurotoxicity of the amyloid- peptides on their potentially negative interaction with neuronal membranes. It is known that amyloid-ß peptides interact with the membrane, modifying the membrane's structural and dynamic properties. We present a series of X-ray diffraction experiments on anionic model lipid membranes containing various amounts of cholesterol. These experiments provide experimental evidence for an interaction of both the full length amyloid-ß1-42 peptide, and the peptide fragment amyloid-ß22-40 with anionic bilayer containing cholesterol. The location of the amyloid-ß peptides was determined from these experiments, with the full length peptide embedding into the membrane, and the peptide fragment occupying 2 positions-on the membrane surface and embedded into the membrane core.

Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations.

Understanding sugar-lipid interactions during desiccation and freezing is an important step in the elucidation of cryo- and anhydro-protection mechanisms. We determine sucrose, trehalose, and water concentration distributions in intra-bilayer volumes between opposing dioleoylphosphatidylcholine bilayers over a range of reduced hydrations and sugar concentrations. Stacked lipid bilayers at reduced hydration provide a suitable system to mimic environmental dehydration effects, as well as a suitable system for direct probing of sugar locations by neutron membrane diffraction. Sugar distributions show that sucrose and trehalose both behave as typical uncharged solutes, largely excluded from the lipid bilayers regardless of sugar identity, and with no correlation between sugar distribution and the lipid headgroup position as the hydration is changed. These results are discussed in terms of current opinions about cryo- and anhydro-protection mechanisms.

Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms.

Trehalose, a natural disaccharide with bioprotective properties, is widely recognized for its ability to preserve biological membranes during freezing and dehydration events. Despite debate over the molecular mechanisms by which this is achieved, and that different mechanisms imply quite different distributions of trehalose molecules with respect to the bilayer, there are no direct experimental data describing the location of trehalose within lipid bilayer membrane systems during dehydration. Here, we use neutron membrane diffraction to conclusively show that the trehalose distribution in a dioleoylphosphatidylcholine (DOPC) system follows a Gaussian profile centred in the water layer between bilayers. The absence of any preference for localizing near the lipid headgroups of the bilayers indicates that the bioprotective effects of trehalose at physiologically relevant concentrations are the result of non-specific mechanisms that do not rely on direct interactions with the lipid headgroups.

Neutron diffraction studies of oral stratum corneum model lipid membranes.

In this work we have investigated model lipid mixtures simulating a lipid component of oral stratum corneum (OSC). Neutron diffraction experiments on oriented samples have revealed that SM (bovine brain)/dipalmitoylphosphatidylethanolamine/dipalmitoylphosphatidylcholine (DPPE/DPPC) mixtures at molar ratios of 1/2/1 and 1/1/1 are one-phase membranes. The incorporation of low concentrations of ceramide 6 and cholesterol into SM/DPPC/DPPE bilayers does not result in a phase separation, affecting membrane hydration. The model OSC membrane composed of ceramide 6/cholesterol/fatty acids/cholesterol sulfate/SM (bovine brain)/DPPE/DPPC is characterized by coexistence of several lamellar phases, that behave differently during their hydration in water excess. The phase with lamellar repeat distance of about 45 Å is likely a ceramide-rich phase and shows a restricted swelling in water, while another phase with repeat distance of 50 Å swells very quickly on 15 Å and then disappears. Our results indicate that phospholipid-rich and ceramide-rich domains could possibly coexist in the intercellular space of oral epithelium.

CO2 sorption to subsingle hydration layer montmorillonite clay studied by excess sorption and neutron diffraction measurements.

Geologic storage of CO(2) requires that the caprock sealing the storage rock is highly impermeable to CO(2). Swelling clays, which are important components of caprocks, may interact with CO(2) leading to volume change and potentially impacting the seal quality. The interactions of supercritical (sc) CO(2) with Na saturated montmorillonite clay containing a subsingle layer of water in the interlayer region have been studied by sorption and neutron diffraction techniques. The excess sorption isotherms show maxima at bulk CO(2) densities of ≈ 0.15 g/cm(3), followed by an approximately linear decrease of excess sorption to zero and negative values with increasing CO(2) bulk density. Neutron diffraction experiments on the same clay sample measured interlayer spacing and composition. The results show that limited amounts of CO(2) are sorbed into the interlayer region, leading to depression of the interlayer peak intensity and an increase of the d(001) spacing by ca. 0.5 Å. The density of CO(2) in the clay pores is relatively stable over a wide range of CO(2) pressures at a given temperature, indicating the formation of a clay-CO(2) phase. At the excess sorption maximum, increasing CO(2) sorption with decreasing temperature is observed while the high-pressure sorption properties exhibit weak temperature dependence.

Lipophilic penetration enhancers and their impact to the bilayer structure of stratum corneum lipid model membranes: neutron diffraction studies based on the example oleic acid.

The present study analyzes the effect of the lipophilic penetration enhancer oleic acid on the bilayer structure of stratum corneum (SC) lipid model membranes based on Ceramide AP by using the neutron diffraction technique. Our results indicate the formation of a single lamellar phase in the presence of oleic acid under the chosen experimental conditions; a separated fluid-like oleic acid-rich phase was not detected in the present study. By comparing the internal membrane structure received from Fourier synthesis with the model system lacking oleic acid, considerable structural changes in terms of impairment of the lamellar order were found after incorporation of the penetration enhancer into the bilayers. In addition, by using specifically deuterated oleic acid we were able to prove the integration of the enhancer molecules into the model bilayers and moreover, to determine the exact position of oleic acid inside the SC lipid model membrane. From the present results we conclude a strong perturbation of lamellar SC lipid arrangement due to the intercalated penetration enhancer which can account for the promoting effects on drug penetration across the SC known for oleic acid.