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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

Nanoscale structural and mechanical effects of beta-amyloid (1-42) on polymer cushioned membranes: a combined study by neutron reflectometry and AFM Force Spectroscopy.

The interaction of beta-amyloid peptides with lipid membranes is widely studied as trigger agents in Alzheimer's disease. Their mechanism of action at the molecular level is unknown and their interaction with the neural membrane is crucial to elucidate the onset of the disease. In this study we have investigated the interaction of water soluble forms of beta-amyloid Aß(1-42) with lipid bilayers supported by polymer cushion. A reproducible protocol for the preparation of a supported phospholipid membrane with composition mimicking the neural membrane and in physiological condition (PBS buffer, pH=7.4) was refined by neutron reflectivity. The change in structure and local mechanical properties of the membrane in the presence of Aß(1-42) was investigated by neutron reflectivity and Atomic Force Microscopy (AFM) Force Spectroscopy. Neutron reflectivity evidenced that Aß(1-42) interacts strongly with the supported membrane, causing a change in the scattering length density profile of the lipid bilayer, and penetrates into the membrane. Concomitantly, the local mechanical properties of the bilayer are deeply modified by the interaction with the peptide as seen by AFM Force Spectroscopy. These results may be of great importance for the onset of the Alzheimer's disease, since a simultaneous change in the structural and mechanical properties of the lipid matrix could influence all membrane based signal cascades.

Alzheimer's disease amyloid-beta peptide analogue alters the ps-dynamics of phospholipid membranes.

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-beta (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-beta (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290K and 320K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-beta (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-beta (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-beta (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-beta (25-35) peptide induced membrane alteration even at only 3mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.

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.

Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling.

This research paper provides direct evidence concerning the localisation of free fatty acids in stratum corneum lipid model membranes. We employed partially deuterated free fatty acids to gain further information about the assembly of a stratum corneum lipid model membrane based on a ceramide of the phytosphingosine-type (ceramide [AP]) with particular respect to the position of the deuterated groups of the free fatty acids. The application of behenic-22,22,22-d(3)-acid and cerotic-12,12,13,13-d(4)-acid confirmed that the short-chain ceramide [AP] forces the longer-chained free fatty acids to incorporate into the bilayer created by ceramide [AP]. The ceramide [AP] molecules determine the structural assembly of this model membrane and obligate the long-chain free fatty acids to either arrange inside this formation or to separate as a fatty acid rich phase.

Basic nanostructure of stratum corneum lipid matrices based on ceramides [EOS] and [AP]: a neutron diffraction study.

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured omega-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of approximately 1 A, consequently the headgroups of the neighboring leaflets are positioned close to each other.

Analysis of microporosity in ordered mesoporous hierarchically structured silica by combining physisorption with in situ small-angle scattering (SAXS and SANS).

The combination of physisorption experiments with simultaneous in situ small-angle X-ray and neutron scattering (SAXS/SANS) was used to elucidate the porosity in mesoporous silica with a trimodal pore structure. The material ("KLE-IL") contains spherical mesopores of 14 nm in diameter, worm-like mesopores (2-3 nm), and micropores, templated by a block copolymer and an ionic liquid surfactant, while the micropores originate from the hydrophilic block of the block copolymer. The main objective of the study was the quantification of the microporosity and the small mesopores and to find out if they are indeed located between the larger, spherical mesopores. Our in situ SAXS/SANS experiments took advantage of contrast matching of nitrogen (SANS, T = 77 K) and dibromomethane (SAXS, T = 290 K). By using the latter gas with a slightly larger kinetic diameter, it was possible to judge the accessibility of the pores under ambient conditions. The in situ experiments were supported by high-precision ex situ physisorption. Using suitable approaches for the SAXS/SANS analysis, it was possible to separate the content of the micropores and small mesopores.