Microscopic diffusion processes measured in living planarians

Living planarian flatworms were probed using quasielastic neutron scattering to measure, on the pico-to-nanosecond time scale and nanometer length scale, microscopic diffusion of water and cell constituents in the planarians. Measurable microscopic diffusivities were surprisingly well defined in such a complex system as living animals. The overall variation in the microscopic diffusivity of cell constituents was found to be far lower than the variation in the microscopic diffusivity of water in planarians in a temperature range of 284.5 to 304.1 K.

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title: Down to atomic‐scale intracellular water dynamics
abstract: Water constitutes the intracellular matrix in which biological molecules interact. Understanding its dynamic state is a main scientific challenge, which continues to provoke controversy after more than 50 years of study. We measured water dynamics in vivo in the cytoplasm of Escherichia coli by using neutron scattering and isotope labelling. Experimental timescales covered motions from pure water to interfacial water, on an atomic length scale. In contrast to the widespread opinion that water is ‘tamed’ by macromolecular confinement, the measurements established that water diffusion within the bacteria is similar to that of pure water at physiological temperature.

title: Intracellular water - an overlooked drug target? Cisplatin impact in cancer cells probed by neutrons.
abstract: The first neutron scattering study on human nucleated cells is reported, addressing the subject of solvent-slaving to a drug by probing intracellular water upon drug exposure. Inelastic and quasi-elastic neutron scattering spectroscopy with isotope labelling was applied for monitoring interfacial water response to the anticancer drug cisplatin, in the low prognosis human metastatic breast cancer cells MDA-MB-231. Optical vibrational data were also obtained for lyophilised cells. Concentration-dependent dynamical changes evidencing a progressive mobility reduction were unveiled between untreated and cisplatin-exposed samples, concurrent with variations in the native organisation of water molecules within the intracellular medium as a consequence of drug action. The results thus obtained yielded a clear picture of the intracellular water response to cisplatin and constitute the first reported experimental proof of a drug impact on the cytomatrix by neutron techniques. This is an innovative way of tackling a drug's pharmacodynamics, searching for alternative targets of drug action.

title: In vivo measurement of internal and global macromolecular motions in Escherichia coli.
abstract: We present direct quasielastic neutron scattering measurements, in vivo, of macromolecular dynamics in Escherichia coli. The experiments were performed on a wide range of timescales to cover the large panel of internal and self-diffusion motions. Three major internal processes were extracted at physiological temperature: a fast picosecond process that corresponded to restricted jump diffusion motions and two slower processes that resulted from reorientational motions occurring in approximately 40 ps and 90 ps, respectively. The analysis of the fast process revealed that the cellular environment leads to an appreciable increase in internal molecular flexibility and diffusive motion rates compared with those evaluated in fully hydrated powders. The result showed that the amount of cell water plays a decisive role in internal molecular dynamics. Macromolecular interactions and confinement, however, attenuate slightly the lubricating effect of water, as revealed by the decrease of the in vivo parameters compared with those measured in solution. The study demonstrated that standard sample preparations do not mimic accurately the physiological environment and suggested that intracellular complexity participates in functional dynamics necessary for biological activity. Furthermore, the method allowed the extraction of the self-diffusion of E. coli macromolecules, which presented similar parameters as those extracted for hemoglobin in red blood cells.

title: Hemoglobin dynamics in red blood cells: correlation to body temperature.
abstract: A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

title: Neutron scattering: a tool to detect in vivo thermal stress effects at the molecular dynamics level in micro-organisms
abstract: In vivo molecular dynamics in Halobacterium salinarum cells under stress conditions was measured by neutron scattering experiments coupled with microbiological characterization. Molecular dynamics alterations were detected with respect to unstressed cells, reflecting a softening of protein structures consistent with denaturation. The experiments indicated that the neutron scattering method provides a promising tool to study molecular dynamics modifications in the proteome of living cells induced by factors altering protein folds.

title: Deep Sea Microbes Probed by Incoherent Neutron Scattering Under High Hydrostatic Pressure
abstract: The majority of the biosphere is a high pressure environment. Around 70% of the marine biosphere lies at depths below 1000 m, i.e. at pressures of 100 bars or higher. To survive in these environments, deep-biosphere organisms have adapted to life at high pressure. In vitro studies showed that the activity of certain proteins originating from deep-sea organisms is less affected by high pressure than that of enzymes from surface organisms . However, the genetic and structural bases for this increased pressure resistance are still unknown. Elastic incoherent neutron scattering studies, which provide access to information about molecular dynamics, constitute a very promising approach to decipher the structural adaptation in proteins living under high pressure. This approach has been used in the past to investigate the adaptation of biological systems to temperature and salinity and proved to be essential and complementary to structural studies. Here first investigations of high pressure effects on cell dynamics are presented using Thermococcales as models.

title: Molecular adaptation and salt stress response of Halobacterium salinarum cells revealed by neutron spectroscopy
abstract: Halobacterium salinarum is an extreme halophile archaeon with an absolute requirement for a multimolar salt environment. It accumulates molar concentrations of KCl in the cytosol to counterbalance the external osmotic pressure imposed by the molar NaCl. As a consequence, cytosolic proteins are permanently exposed to low water activity and highly ionic conditions. In non-adapted systems, such conditions would promote protein aggregation, precipitation, and denaturation. In contrast, in vitro studies showed that proteins from extreme halophilic cells are themselves obligate halophiles. In this paper, adaptation via dynamics to low-salt stress in H. salinarum cells was measured by neutron scattering experiments coupled with microbiological characterization. The molecular dynamic properties of a proteome represent a good indicator for environmental adaptation and the neutron/microbiology approach has been shown to be well tailored to characterize these modifications. In their natural setting, halophilic organisms often have to face important variations in environmental salt concentration. The results showed deleterious effects already occur in the H. salinarum proteome, even when the external salt concentration is still relatively high, suggesting the onset of survival mechanisms quite early when the environmental salt concentration decreases.

title: High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes
abstract: Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

title: Specific cellular water dynamics observed in vivo by neutron scattering and NMR.
abstract: Neutron scattering, by using deuterium labelling, revealed how intracellular water dynamics, measured in vivo in E. coli, human red blood cells and the extreme halophile, Haloarcula marismortui, depends on the cell type and nature of the cytoplasm. The method uniquely permits the determination of motions on the molecular length (approximately ångstrøm) and time (pico- to nanosecond) scales. In the bacterial and human cells, intracellular water beyond the hydration shells of cytoplasmic macromolecules and membrane faces flows as freely as liquid water. It is not "tamed" by confinement. In contrast, in the extreme halophile archaeon, in addition to free and hydration water an intracellular water component was observed with significantly slowed down translational diffusion. The results are discussed and compared to observations in E. coli and Haloarcula marismortui by deuteron spin relaxation in NMR--a method that is sensitive to water rotational dynamics on a wide range of time scales.

title: Organization and Flexibility of Cyanobacterial Thylakoid Membranes Examined by Neutron Scattering
abstract: In cyanobacteria, light harvesting and photosynthesis occur in the thylakoid membranes.  The distances between thylakoid membranes are correlated with the size of the phycobilisome antenna and change reversibly and rapidly upon illumination.  Thylakoid membranes have a structural plasticity tied to the regulation of photosynthesis.  Characterizing the structural changes in photosynthetic membranes is crucial for understanding light harvesting and photosynthetic productivity. Cyanobacteria are prokaryotes that can use photosynthesis to convert sunlight into cellular fuel. Knowledge of the organization of the membrane systems in cyanobacteria is critical to understanding the metabolic processes in these organisms. We examined the wild-type strain of Synechocystis sp. PCC 6803 and a series of mutants with altered light-harvesting phycobilisome antenna systems for changes in thylakoid membrane architecture under different conditions. Using small-angle neutron scattering, it was possible to resolve correlation distances of subcellular structures in live cells on the nanometer scale and capture dynamic light-induced changes to these distances. Measurements made from samples with varied scattering contrasts confirmed that these distances could be attributed to the thylakoid lamellar system. We found that the changes to the thylakoid system were reversible between light- and dark-adapted states, demonstrating a robust structural flexibility in the architecture of cyanobacterial cells. Chemical disruption of photosynthetic electron transfer diminished these changes, confirming the involvement of the photosynthetic apparatus. We have correlated these findings with electron microscopy data to understand the origin of the changes in the membranes and found that light induces an expansion in the center-to-center distances between the thylakoid membrane layers. These combined data lend a dynamic dimension to the intracellular organization in cyanobacterial cells.

title: Fast internal dynamics in alcohol dehydrogenase.
abstract: Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

title: Multiple-scattering effects on smooth neutron-scattering spectra
abstract: Elastic and inelastic incoherent neutron-scattering experiments are simulated for simple models: a rigid solid (as used for normalization), a glass (with a smooth distribution of harmonic vibrations), and a viscous liquid (described by schematic mode-coupling equations). In cases where the input scattering law factorizes into a wave-number-dependent amplitude and a frequency-dependent spectral distribution, the latter is only weakly affected by multiple scattering, whereas the former is severely distorted.

title: Quasi-elastic neutron scattering in the multi-GPa range and its application to liquid water
abstract: We present a method which allows quasi-elastic neutron scattering (QENS) at multi-GPa pressures, i.e., pressure conditions which are an order of magnitude higher than possible with QENS till now. The technique uses an opposed-anvil geometry with a sample chamber in the form of a toroid which minimizes multiple scattering effects. This method has been applied to measurements of the self-diffusion coefficient of water at 400 K up to 3 GPa. The results demonstrate the potential of this method for studying diffusion over a large pressure and temperature range.

title: Intracellular Water Exchange for Measuring the Dry Mass, Water Mass and Changes in Chemical Composition of Living Cells
abstract: We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell’s buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell’s water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell’s dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density – the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

title: Evolution of the internal dynamics of two globular proteins from dry powder to solution.
abstract: Myoglobin and lysozyme picosecond internal dynamics in solution is compared to that in hydrated powders by quasielastic incoherent neutron scattering. This technique is sensitive to the motions of the nonexchangeable hydrogen atoms in a sample. Because these are homogeneously distributed throughout the protein structure, the average dynamics of the protein is described. We first propose an original data treatment to deal with the protein global motions in the case of solution samples. The validity of this treatment is checked by comparison with classical measurements of the diffusion constants. The evolution with the scattering vector of the width and relative contribution of the quasielastic component was then used to derive information on the amount of local diffusive motions and their characteristic average relaxation time. From dry powder to coverage by one water layer, the surface side chains progressively acquire the possibility to diffuse locally. On subsequent hydration, the main effect of water is to improve the rate of these diffusive motions. Motions with higher average amplitude occur in solution, about three times more than for a hydrated powder at complete coverage, with a shorter average relaxation time, approximately 4.5 ps compared to 9.4 ps for one water monolayer.

title: Dynamics of well-folded and natively disordered proteins in solution: a time-of-flight neutron scattering study
abstract: Casein proteins belong to the class of natively disordered proteins. The existence of disordered biologically active proteins questions the assumption that a well-folded structure is required for function. A hypothesis generally put forward is that the unstructured nature of these proteins results from the functional need of a higher flexibility. This interplay between structure and dynamics was investigated in a series of time-of-flight neutron scattering experiments, performed on casein proteins, as well as on three well-folded proteins with distinct secondary structures, namely, myoglobin (α), lysozyme (α/β) and concanavalin A (β). To illustrate the subtraction of the solvent contribution from the scattering spectra, we used the dynamic susceptibility spectra emphasizing the high frequency part of the spectrum, where the solvent dominates. The quality of the procedure is checked by comparing the corrected spectra to those of the dry and hydrated protein with negligible solvent contamination. Results of spectra analysis reveal differences in motional amplitudes of well-folded proteins, where β-sheet structures appear to be more rigid than a cluster of α-helices. The disordered caseins display the largest conformational displacements. Moreover their global diffusion rates deviate from the expected dependence, suggesting further large-scale conformational motions.

title: Protein diffusion in crowded electrolyte solutions.
abstract: We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.

title: Protein self-diffusion in crowded solutions
abstract: Macromolecular crowding in biological media is an essential factor for cellular function. The interplay of intermolecular interactions at multiple time and length scales governs a fine-tuned system of reaction and transport processes, including particularly protein diffusion as a limiting or driving factor. Using quasielastic neutron backscattering, we probe the protein self-diffusion in crowded aqueous solutions of bovine serum albumin on nanosecond time and nanometer length scales employing the same protein as crowding agent. The measured diffusion coefficient D(φ) strongly decreases with increasing protein volume fraction φ explored within 7% ≤ φ ≤ 30%. With an ellipsoidal protein model and an analytical framework involving colloid diffusion theory, we separate the rotational Dr(φ) and translational Dt(φ) contributions to D(φ). The resulting Dt(φ) is described by short-time self-diffusion of effective spheres. Protein self-diffusion at biological volume fractions is found to be slowed down to 20% of the dilute limit solely due to hydrodynamic interactions.

title: Dynamics of highly concentrated protein solutions around the denaturing transition
abstract: Using both quasi-elastic and fixed-window neutron spectroscopy, we study the dynamics of highly concentrated aqueous protein solutions of bovine serum albumin around the denaturing transition. For the temperature range 280 K < T < 370 K, the total mean-squared displacement 〈u2〉 is recorded. Below and above the denaturing, we observe that 〈u2〉 increases monotonically with T, but at the denaturing transition it decreases strongly. This observation can be rationalized and quantitatively modeled as a transition from a liquid protein solution to a gel-like state. Atomic vibrations, molecular subunit diffusion and, most importantly, diffusion of the entire protein determine 〈u2〉. The latter is strongly hindered due to entanglement and cross-linking of the chains and causes the pronounced decrease of 〈u2〉. Using information from the full quasi-elastic signal, we separate the diffusion contribution from 〈u2〉 and reveal the transition temperature. For the analysis of this separation, we introduce a general concept, which is applicable to other colloid systems exhibiting both center-of-mass and internal dynamics.

title: Diffusion and dynamics of γ-globulin in crowded aqueous solutions.
abstract: Dynamics in protein solutions is essential for both protein function and cellular processes. The hierarchical complexity of global protein diffusion, side-chain diffusion, and microscopic motions of chemical groups renders a complete understanding challenging. We present results from quasi-elastic neutron scattering on protein solutions of γ-globulin over a wide range of volume fractions. Translational and rotational diffusion can be self-consistently separated from internal motions. The global diffusion is consistent with predictions for effective spheres even though the branched molecular shape differs considerably from a colloidal sphere. The internal motions are characterized both geometrically and dynamically, suggesting a picture of methyl rotations and restricted diffusion of side chains. We show that the advent of new neutron spectrometers allows the study of current questions including the coupling of intracellular dynamics and protein function.

title: Hierarchical molecular dynamics of bovine serum albumin in concentrated aqueous solution below and above thermal denaturation.
abstract: The dynamics of proteins in solution is a complex and hierarchical process, affected by the aqueous environment as well as temperature. We present a comprehensive study on nanosecond time and nanometer length scales below, at, and above the denaturation temperature Td. Our experimental data evidence dynamical processes in protein solutions on three distinct time scales. We suggest a consistent physical picture of hierarchical protein dynamics: (i) self-diffusion of the entire protein molecule is confirmed to agree with colloid theory for all temperatures where the protein is in its native conformational state. At higher temperatures T > Td, the self-diffusion is strongly obstructed by cross-linking or entanglement. (ii) The amplitude of backbone fluctuations grows with increasing T, and a transition in its dynamics is observed above Td. (iii) The number of mobile side-chains increases sharply at Td while their average dynamics exhibits only little variations. The combination of quasi-elastic neutron scattering and the presented analytical framework provides a detailed microscopic picture of the protein molecular dynamics in solution, thereby reflecting the changes of macroscopic properties such as cluster formation and gelation.

title: Salt-Induced Universal Slowing Down of the Short-Time Self-Diffusion of a Globular Protein in Aqueous Solution.
abstract: The short-time self-diffusion D of the globular model protein bovine serum albumin in aqueous (D2O) solutions has been measured comprehensively as a function of the protein and trivalent salt (YCl3) concentration, noted cp and cs, respectively. We observe that D follows a universal master curve D(cs,cp) = D(cs = 0,cp) g(cs/cp), where D(cs = 0,cp) is the diffusion coefficient in the absence of salt and g(cs/cp) is a scalar function solely depending on the ratio of the salt and protein concentration. This observation is consistent with a universal scaling of the bonding probability in a picture of cluster formation of patchy particles. The finding corroborates the predictive power of the description of proteins as colloids with distinct attractive ion-activated surface patches.

title: Nanoscopic dynamics of phospholipid in unilamellar vesicles: effect of gel to fluid phase transition.
abstract: The dynamics of phospholipids in unilamellar vesicles (ULVs) is of interest in biology, medical, and food sciences, since these molecules are widely used as biocompatible agents and a mimic of cell membrane systems. We have investigated the nanoscopic dynamics of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid in ULVs as a function of temperature using elastic and quasielastic neutron scattering (QENS). The dependence of the signal on the scattering momentum transfer, which is a critical advantage of neutron scattering techniques, allows the detailed analysis of the lipid motions that cannot be carried out by other means. In agreement with a differential scanning calorimetry measurement, a sharp rise in the elastic scattering intensity below ca. 296 K indicates a phase transition from the high-temperature fluid phase to the low-temperature solid gel phase. The microscopic lipid dynamics exhibits qualitative differences between the solid gel phase (in a measurement at 280 K) and the fluid phase (in a measurement at a physiological temperature of 310 K). The analysis of the data demonstrates the presence of two types of distinct motions: the entire lipid molecule motion within a monolayer, also known as lateral diffusion, and the relatively faster internal motion of the DMPC molecule. The lateral diffusion of the entire lipid molecule is Fickian in character, whereas the internal lipid motions are of localized character, which is consistent with the structure of the vesicles. The lateral motion slows down by an order of magnitude in the solid gel phase, whereas for the internal motion not only the time scale but also the character of the motion changes upon the phase transition. In the solid gel phase, the lipids are more ordered and undergo uniaxial rotational motion. However, in the fluid phase, the hydrogen atoms of the lipid tails undergo confined translation diffusion rather than uniaxial rotational diffusion. The translational, but spatially localized, diffusion of the hydrogen atoms of the lipid tails is a manifestation of the flexibility of the chains acquired in the fluid phase. Because of this flexibility, both the local diffusivity and the confinement volume for the hydrogen atoms increase in the linear fashion from near the lipid's polar headgroup to the end of its hydrophobic tail. Our results present a quantitative and detailed picture of the effect of the gel-fluid phase transition on the nanoscopic lipid dynamics in ULVs. The data analysis approach developed here has a potential for probing the dynamic response of lipids to the presence of additional cell membrane components.

title: Effect of antimicrobial peptide on the dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer.
abstract: Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fall in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol% melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Their importance lies in the fact that many membrane processes, including signaling and energy transduction pathways, are controlled to a great extent by the lateral diffusion of lipids in the membrane. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with cholesterol in the presence and absence of 0.2 mol% melittin. Remarkably, the effects of melittin on the membrane dynamics disappear in the presence of 20 mol% cholesterol. Our measurements indicate that the destabilizing effect of the peptide melittin on membranes can be mitigated by the presence of cholesterol. This study might provide new insights into the mechanism of action of antimicrobial peptides and their selective toxicity towards foreign microorganisms.

title: Effect of α-Tocopherol on the Microscopic Dynamics of Dimyristoylphosphatidylcholine Membrane.
abstract: Vitamin E behaves as an antioxidant and is well known for its protective properties of the lipid membrane. The most biologically active form of vitamin E in the human organism is α-tocopherol (aToc). Very recently (Marquardt, D.; et al.  2014, 136, 203-210) it has been shown that aToc resides near the center of dimyristoylphosphatidylcholine (DMPC) bilayer, which is in stark contrast with other PC membranes, where aToc is located near the lipid-water interface. Here we report an unusual effect of this exceptional location of aToc on the dynamical behavior of DMPC membrane probed by incoherent elastic and quasielastic neutron scattering. For pure DMPC vesicles, elastic scan data show two step-like drops in the elastic intensity at 288 and 297 K, which correspond to the pre- and main phase transitions, respectively. However, inclusion of aToc into DMPC membrane inhibits the step-like elastic intensity drops, indicating a significant impact of aToc on the phase behavior of the membrane. This observation is supported by our differential scanning calorimetry data, which shows that inclusion of aToc leads to a significant broadening of the main phase transition peak, whereas the peak corresponding to the pretransition disappears. We have performed quasielastic neutron scattering (QENS) measurements on DMPC vesicles with various concentrations of aToc at 280, 293, and 310 K. We have found that aToc affects both the lateral diffusion and the internal motions of the lipid molecules. Below the main phase transition temperature inclusion of aToc accelerates both the lateral and the internal lipid motions. On the other hand, above the main phase transition temperature the addition of aToc restricts only the internal motion, without a significant influence on the lateral motion. Our results support the finding that the location of aToc in DMPC membrane is deep within the bilayer.

title: Dynamical and Phase Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low Concentration.
abstract: The mechanism of action of antimicrobial peptides is traditionally attributed to the formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert a similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature no longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides.

title: Water-protein dynamic coupling and new opportunities for probing it at low to physiological temperatures in aqueous solutions.
abstract: Both the structure and dynamics of biomolecules are known to be essential for their biological function. In the dehydrated state, the function of biomolecules, such as proteins, is severely impeded, so hydration is required for bioactivity. The dynamics of the hydrated biomolecules and their hydration water are related - but how closely? The problem involves several layers of complexity. Even for water in the bulk state, the contribution from various dynamic components to the overall dynamics is not fully understood. In biological systems, the effects of confinement on the hydration water further complicate the picture. Even if the various components of the hydration water dynamics are properly understood, which of them are coupled to the protein dynamics, and how? The studies of protein dynamics over the wide temperature range, from physiological to low temperatures, provide some answers to these question. At low temperatures, both the protein and its hydration water behave as solids, with only vibrational degrees of freedom. As the temperature is increased, non-vibrational dynamic components start contributing to the measurable dynamics and eventually become dominant at physiological temperatures. Thus, the temperature dependence of the dynamics of protein and its hydration water may allow probing various dynamic components separately. In order to suppress the water freezing, the low-temperature studies of protein rely on either low-hydrated samples (essentially, hydrated protein powders), or cryo-protective solutions. Both approaches introduce the hydration environments not characteristic of the protein environments in living systems, which are typically aqueous protein solutions of various concentrations. In this paper, we discuss the coupling between the dynamic components of the protein and its hydration water by critical examining of the existing literature, and then propose that proteins can be studied in an aqueous solution that is remarkably similar in its dynamic properties to pure water, yet does not freeze down to about 200 K, even in the bulk form. The first experiment of this kind using quasielastic neutron scattering is discussed, and more experiments are proposed.

title: Low-density lipoproteins investigated under high hydrostatic pressure by elastic incoherent neutron scattering
abstract: Human low-density lipoprotein (LDL) is a highly complex nano-particle built up of various lipid classes and a single large protein moiety (apoB-100) owning essential physiological functions in the human body. Besides its vital role as a supplier of cholesterol and fat for peripheral tissues and cells, it is also a known key player in the formation of atherosclerosis. Due to these important roles in physiology and pathology the elucidation of structural and dynamical details is of great interest. In the current study we drew a broader picture of LDL dynamics using elastic incoherent neutron scattering (EINS) as a function of specified temperature and pressure points. We not only investigated a normolipidemic LDL sample, but also a triglyceride-rich and an oxidized one to mimic pathologic conditions as found under hyperlipidemic conditions or in atherosclerotic plaques, respectively. We could show that pressure has a significant effect on atomic motions in modified forms of LDL, whereas the normolipidemic sample seems to cope much better with high-pressure conditions irrespective of temperature. These findings might be explained by the altered lipid composition, which is either caused through elevated triglyceride content or modifications through lipid peroxidation.

title: Differential scanning calorimetry of porcine adipose tissues.
abstract: Differential scanning calorimetry (DSC) was used for the direct analysis of melting properties in porcine subcutaneous, intermuscular, and kidney leaf adipose tissue by heating at a constant ratio of +5°C/min from 4 to 90°C. Melting curves for adipose tissues as well as fat extracted from the associated tissues by chloroform-methanol were generated using DSC. Major peaks in DSC curves were similar among types of adipose tissue but the temperatures of the melting peak and conclusion point differed among types of adipose tissues. From the visual appearance of fat samples it appeared that the major DSC peak corresponded to phase transition of the fat. The direct DSC analysis of porcine adipose tissues may be useful to determine melting properties.

title: A time-of-flight backscattering spectrometer at the Spallation Neutron Source, BASIS.
abstract: We describe the design and current performance of the backscattering silicon spectrometer (BASIS), a time-of-flight backscattering spectrometer built at the spallation neutron source (SNS) of the Oak Ridge National Laboratory (ORNL). BASIS is the first silicon-based backscattering spectrometer installed at a spallation neutron source. In addition to high intensity, it offers a high-energy resolution of about 3.5 μeV and a large and variable energy transfer range. These ensure an excellent overlap with the dynamic ranges accessible at other inelastic spectrometers at the SNS.