| 1. |
- Björling, Alexander, 1983, et al.
(author)
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Ubiquitous Structural Signaling in Bacterial Phytochromes
- 2015
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In: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 6:17, s. 3379-3383
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Journal article (peer-reviewed)abstract
- The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.
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| 2. |
- Brändén, Gisela, 1975, et al.
(author)
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Coherent diffractive imaging of microtubules using an X-ray laser.
- 2019
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In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1
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Journal article (peer-reviewed)abstract
- X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.
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| 3. |
- Dods, Robert, 1989, et al.
(author)
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From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.
- 2017
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In: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 25:9, s. 1461-1468
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Journal article (peer-reviewed)abstract
- Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.
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| 4. |
- Dods, Robert, 1989, et al.
(author)
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Ultrafast structural changes within a photosynthetic reaction centre.
- 2021
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In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 589:7841, s. 310-314
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Journal article (peer-reviewed)abstract
- Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
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| 5. |
- Arnlund, David, et al.
(author)
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Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser
- 2014
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In: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:9, s. 923-926
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Journal article (peer-reviewed)abstract
- We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
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| 6. |
- Berntsen, Peter, 1974, et al.
(author)
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Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells
- 2010
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In: Journal of the Royal Society Interface. - : The Royal Society. - 1742-5689 .- 1742-5662. ; 7:Suppl 3
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Journal article (peer-reviewed)abstract
- The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 mu m) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 mu m), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 mu M did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.
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| 7. |
- Berntsen, Peter, 1974, et al.
(author)
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Dielectric and calorimetric studies of hydrated purple membrane
- 2005
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 89:5, s. 3111-3128
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Journal article (peer-reviewed)abstract
- Purple membranes (PM) from halobacteria were hydrated to ∼0.4 and ∼0.2 g H 2 O/g of PM and studied by dielectric spectroscopy and differential scanning calorimetry between 120 and 300 K. The dielectric process, attributed to a local (β) relaxation of the confined supercooled water, shows an Arrhenius temperature behavior at low temperatures. In the case of the most hydrated PM a small deviation from the Arrhenius behavior occurs at 190-200 K together with a pronounced endothermic process and an increased activation energy. The observed crossover is accompanied by a reduction of the interlayer spacing due to the partial loss of the intermembrane water. All these effects at ∼200 K are consistent with a scenario where the local relaxation process merges with a nonobservable α-relaxation of the interlayer water, giving rise to a more liquid-like behavior of the interfacial water. For the less hydrated sample the effects are less pronounced and shift to a slightly higher temperature. © 2005 by the Biophysical Society.
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| 8. |
- Berntsen, Peter, 1974
(author)
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Dynamics of biological membranes and associated water.
- 2008
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Licentiate thesis (other academic/artistic)abstract
- An ingenious assembly of biomolecules and water constitute what we call biological tissue. The presence of water in this assembly is fundamental for the physiological processes and can make the difference between life and death. This thesis is concerned with fundamental questions related to the dynamics of water and its presence around all living cells. The cell is surrounded by a membrane that not only serve as a covering, within which the cell can function, but also by means of transport for life essential species. The capacity of membrane to cooperate with its surrounding is life essential and made possible by to proteins and polar lipids, essentially phospholipids that make the membranes soft. Phospholipids are so called amphipathic molecules, i.e. they have a hydrophobic part (repels water) and a hydrophilic (water loving) part. The hydrophilic region is usually referred to as the head group, and the hydrophobic part is known as the tails. The amphipathic character of the phospholipidsenable them to form lamellar structures called bilayers. In this work hundreds of parallell lipid bilayers on a surface have served as our model system. One bilayer is composed of two layers of lipids arranged so that their hydrocarbon tails face one another, while their charged head groups face the water on either side of the bilayer. We have studied the influence of water in two different membrane systems. One is naturally existing in the plasma membrane of Halobacterium salinarum, which is a bacterium that lives in extremely salty conditions. The other membrane system we have used is from the lecithin group of lipids whose lipid head is made of the alcohol choline (CH3)3N+CH2CH2OH. The lecithins are involved in the transport of other lipids and exist abundantly in the liver and in egg yolk.At biological temperatures there is a mix of many fast and slow motions which makes it diffcult to find out different motions and their possible interrelations. For moderate hydration levels there is no crystallization of the water within the lipid bilayers (not even at temperaures as low as 80K), however there is a substantial slowing-down of the dynamics with decreasing temperature and below a certain temperature the lipids are frozen in a glassy phase. Starting at such low temperatures the onset of different motions of the water and in the lipid systems were probed at successively higher temperatures by using broadband dielectric spectroscopy, differential scanning calorimetry and quasielastic neutron scattering. We found that the molecular motions of the lipids is similar to the molecular motions of glass forming liquids at low temperatures and this behavior was dependent on the amount of water in the lipid system. Transport of charge carriers was also found to be strongly dependent on the water content and this charge transport was changed at the gel-to-liquid transition of the lipids. Furthermore, it was shown that already at 120K (−153◦C) there is local fast motions of the lipid tails, that should be independent of the water content. This work highlights a complex cooperation between water activity and motions in membranes. By shedding light on the coupling between water and membrane motions new insights into the role of water for membrane properties can be gained.
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| 9. |
- Berntsen, Peter, 1974
(author)
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Dynamics of Biomembranes and Rheological Properties of Living Cells
- 2011
-
Doctoral thesis (other academic/artistic)abstract
- An ingenious assembly of biomolecules and water constitute what we call biological tissue. The presence of water in this assembly is fundamental for the physiological processes and can make the difference between life and death. One of the hallmark features of cellular life is the presence of membranes that separates the cell from the rest of the world and its compartments from each other. There is, however, some fluidity through the membranes. This fluid environment is possible due to the presence of water. The close association and dynamical interplay between lipid membranes and the surrounding water is investigated in the first part of this work. Membranes and water constitute important parts in the macromolecular assemblies and architecture that builds the whole living cell. Cells are highly dynamic with internal structures that constantly remodels and respond to external forces like a viscoelastic material. In many critical biological processes, cells both exert and respond to forces in their surroundings; the mechanical and rheological properties of cells are intimately related to their viscoelastic character which is the topic of the second part of this work. At biological temperatures there is a mix of many types of motions occurring on similar time scales which makes it difficult to separate different motions and elucidate interrelations. However, at low temperatures the different dynamical processes occur on considerably more separated time scales, which simplifies the analysis. Starting at low temperatures the onset of different motions of the water and the lipids were probed at successively higher temperatures by using broadband dielectric spectroscopy, differential scanning calorimetry and quasielastic neutron scattering. We found that the molecular motions of the lipids are similar to the molecular motions of glass forming liquids at low temperatures and the exact dynamical behavior was strongly dependent on the amount of water in the lipid system. The hydration water in lipid membranes modulates the motions of the lipid head groups and vice-versa there are also local lipid motions that influence the water dynamics at low hydration levels. Thus, in lipid membranes there is a strong interplay between water and lipid dynamics.The rheological properties of living cells were probed with optical magnetic twisting cytometry. The results show that the cellular mechanical function is susceptible to particulate exposure, and that the structural relaxation time is faster by nearly a factor 10, but also less temperature dependent close to the membrane compared to in the interior cytoskeletal structures. Moreover, for ATP depleted cells the relaxation dynamics slows down around physiological temperatures, which indicates the importance of ATP hydrolysis for the cellular relaxation dynamics.
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| 10. |
- Berntsen, Peter, 1974, et al.
(author)
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Interplay between Hydration Water and Headgroup Dynamics in Lipid Bilayers
- 2011
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 115:8, s. 1825-1832
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Journal article (peer-reviewed)abstract
- In this study, the interplay between water and lipid dynamics has been investigated by broadband dielectric spectroscopy and modulated differential scanning calorimetry (MDSC). The rnultilamellar lipid bilayer system 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) has been studied over a broad temperature range at three different water contents: about 3, 6, and 9 water molecules per lipid molecule. The results from the dielectric relaxation measurements show that at temperatures
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| 11. |
- Berntsen, Peter, 1974, et al.
(author)
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The serial millisecond crystallography instrument at the Australian Synchrotron incorporating the "Lipidico" injector
- 2019
-
In: Review of Scientific Instruments. - : AIP Publishing. - 0034-6748 .- 1089-7623. ; 90:8
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Journal article (peer-reviewed)abstract
- A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an in-house developed high-viscosity injector, "Lipidico." Lipidico uses a syringe needle to extrude the microcrystal-containing viscous media and it is compatible with commercially available syringes. The combination of sample delivery via protein crystals suspended in a viscous mixture and a millisecond frame rate detector enables high-throughput serial crystallography at the Australian Synchrotron. A hit-finding algorithm, based on the principles of "robust-statistics," is employed to rapidly process the data. Here we present the first SMX experimental results with a detector frame rate of 100 Hz (10 ms exposures) and the Lipidico injector using a mixture of lysozyme microcrystals embedded in high vacuum silicon grease. Details of the experimental setup, sample injector, and data analysis pipeline are designed and developed as part of the Australian Synchrotron SMX instrument and are reviewed here. Published under license by AIP Publishing.
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| 12. |
- Nogly, P., et al.
(author)
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Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography
- 2016
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
-
Journal article (peer-reviewed)abstract
- Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
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| 13. |
- Nogly, P., et al.
(author)
-
Lipidic cubic phase serial millisecond crystallography using synchrotron radiation
- 2015
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In: Iucrj. - : International Union of Crystallography (IUCr). - 2052-2525. ; 2
-
Journal article (peer-reviewed)abstract
- Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 angstrom. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
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| 14. |
- Sharma, Amit, et al.
(author)
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A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
-
In: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 0021-8898 .- 1600-5767. ; 52, s. 378-386
-
Journal article (peer-reviewed)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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| 15. |
- Svanberg, Christer, 1970, et al.
(author)
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New class of dynamics in concentrated polymer gels
- 2007
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In: Journal of Chemical Physics. - : AIP Publishing. - 1089-7690 .- 0021-9606. ; 126:5, s. Art. No. 051103-
-
Journal article (peer-reviewed)abstract
- We show that concentrated poly(methyl methacrylate) solution exhibits a new class of coupled dynamics, which can be regarded as an intermediate between the collective diffusion of solutions and the structural relaxations of glasses. This class of dynamics have a relaxation rate that is directly proportional to the wave vector. The transition from diffusive to coupled collective dynamics occurs at smaller length scales with increasing polymer concentration and decreasing temperature. The experimental observations can be understood by considering the contributions from physical cross-links interconnected by stiff polymer segments. © 2007 American Institute of Physics.
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| 16. |
- Svanberg, Christer, 1970, et al.
(author)
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Structural relaxations of phospholipids and water in planar membranes
- 2009
-
In: Journal of Chemical Physics. - : AIP Publishing. - 1089-7690 .- 0021-9606. ; 130:3, s. 035101-
-
Journal article (peer-reviewed)abstract
- We have used dielectric spectroscopy and temperature modulated differential scanning calorimetryTMDSC to investigate the structural relaxation processes and phase transitions of water and lipidsin multilamellar, planar phospholipids. At low hydration levels we observe the main structuralrelaxation related to the glass transition of the phospholipids. With increasing water content a morepronounced pretransition, attributed to a gel to ripple phase transition, is observed in the TMDSCdata. In the proximity of this pretransition, a distinct change in the temperature dependence oralternatively a bifurcation into two processes is observed in the dielectric data. Around thistemperature a crossover in the long-range ionic conductivity across the membranes is also observed,which is one of the key parameters for biological membranes. Thus, the major dynamical changesdo not occur at the main, i.e., the gel to liquid structural phase transition, but at a pretransition thatoccurs roughly 20 K below the main transition.
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| 17. |
- Swenson, Jan, 1966, et al.
(author)
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Solvent and lipid dynamics of hydrated lipid-bilayers by quasielastic neutron scattering
- 2008
-
In: Journal of Chemical Physics. - : AIP Publishing. - 1089-7690 .- 0021-9606. ; 129:4, s. 045101-
-
Journal article (peer-reviewed)abstract
- The microscopic dynamics of the planar, multilamellar lipid bilayer system 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) has been investigated using quasielastic neutron scattering. The DMPC was hydrated to a level corresponding to approximately nine water molecules per lipid molecule. Selective deuteration has been used to separately extract the dynamics of the water, the acyl chains, and the polar head groups from the strong incoherent scattering of the remaining hydrogen atoms. Furthermore, the motions parallel and perpendicular to the bilayers were probed by using two different sample orientations relative to the incident neutron beam. For both sample orientations, the results showed an onset of water motions at 260 K on the experimental time scale of about 100 ps. From lack of wave-vector dependence of the onset temperature for water motions, it is evident that the observed water dynamics is of mainly rotational character at such low temperatures. At 290 K, i.e., slightly below the gel-to-liquid transition around 295 K, the nature of the water dynamics had changed to a more translational character, well described by a jump-diffusion model. On the limited experimental time and length (about 10 Å) scales, this jump-diffusion process was isotropic, despite the very anisotropic system. The acyl chains exhibited a weak onset of anharmonic motions already at 120 K, probably due to conformational changes (trans-gauche and/or syn-anti) in the plane of the lipid bilayers. Other anharmonic motions were not observed on the experimental time scale until temperature had been reached above the gel-to-liquid transition around 295 K, where the acyl chains start to show more substantial motions. © 2008 American Institute of Physics.
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