| 1. |
- Benninger, R. K. P., et al.
(författare)
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Fluorescence imaging of two-photon linear dichroism : Cholesterol depletion disrupts molecular orientation in cell membranes
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:1, s. 609-622
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer ( energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.
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| 2. |
- Lindgren, Mikael, et al.
(författare)
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Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:6, s. 4200-4212
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Tidskriftsartikel (refereegranskat)abstract
- Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300–500 kD) within 2 h that matured after 20 h into larger spherical clusters (30–50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300–500 kD) with an apparent dissociation constant of 1.6 mM, which was slightly better than for ThT (6.8 mM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482nm wavelength when bound to amyloid fibrils.
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| 3. |
- Adler, Jeremy, et al.
(författare)
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Plasma membrane topology and membrane models
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 3:Supplement 1, s. 282a-282a
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 4. |
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| 5. |
- Akanda, Nesar, et al.
(författare)
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Biophysical properties of the apoptosis-inducing plasma membrane voltage-dependent anion channel
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 90:12, s. 4405-4417
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Tidskriftsartikel (refereegranskat)abstract
- Ion channels in the plasma membrane play critical roles in apoptosis. In a recent study we found that a voltage-dependent anion channel in the plasma membrane (VDACpl) of neuronal hippocampal cell line (HT22) cells was activated during apoptosis and that channel block prevented apoptosis. Whether or not VDACpl is identical to the mitochondrial VDACmt has been debated. Here, we biophysically characterize the apoptosis-inducing VDACpl and compare it with other reports of VDACpls and VDACmt. Excised membrane patches of apoptotic HT22 cells were studied with the patch-clamp technique. VDACpl has a large main-conductance state (400 pS) and occasionally subconductance states of µ28 pS and 220 pS. The small subconductance state is associated with long-lived inactivated states, and the large subconductance state is associated with excision of the membrane patch and subsequent activation of the channel. The open-probability curve is bell shaped with its peak around 0mV and is blocked by 30µM Gd3+. The gating can be described by a symmetrical seven-state model with one open state and six closed or inactivated states. These channel properties are similar to those of VDACmt and other VDACpls and are discussed in relation to apoptosis.
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| 6. |
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| 7. |
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| 8. |
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| 9. |
- Andér, Martin, 1979-, et al.
(författare)
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Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 94:3, s. 820-831
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Tidskriftsartikel (refereegranskat)abstract
- The binding of blockers to the human voltage-gated Kv1.5 potassium ion channel is investigated using a three-step procedure consisting of homology modeling, automated docking, and binding free energy calculations from molecular dynamics simulations, in combination with the linear interaction energy method. A reliable homology model of Kv1.5 is constructed using the recently published crystal structure of the Kv1.2 channel as a template. This model is expected to be significantly more accurate than earlier ones based on less similar templates. Using the three-dimensional homology model, a series of blockers with known affinities are docked into the cavity of the ion channel and their free energies of binding are calculated. The predicted binding free energies are in very good agreement with experimental data and the binding is predicted to be mainly achieved through nonpolar interactions, whereas the relatively small differences in the polar contribution determine the specificity. Apart from confirming the importance of residues V505, I508, V512, and V516 for ligand binding in the cavity, the results also show that A509 and P513 contribute significantly to the nonpolar binding interactions. Furthermore, we find that pharmacophore models based only on optimized free ligand conformations may not necessarily capture the geometric features of ligands bound to the channel cavity. The calculations herein give a detailed structural and energetic picture of blocker binding to Kv1.5 and this model should thus be useful for further ligand design efforts.
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| 10. |
- Andersson, Magnus, et al.
(författare)
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A sticky chain model of the elongation and unfolding of escherichia coli P pili under stress
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 90:5, s. 1521-1534
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Tidskriftsartikel (refereegranskat)abstract
- A model of the elongation of P pili expressed by uropathogenic Escherichia coli exposed to stress is presented. The model is based upon the sticky chain concept, which is based upon Hooke’s law for elongation of the layer-to-layer and head-to-tail bonds between neighboring units in the PapA rod and a kinetic description of the opening and closing of bonds, described by rate equations and an energy landscape model. It provides an accurate description of the elongation behavior of P pili under stress and supports a hypothesis that the PapA rod shows all three basic stereotypes of elongation/unfolding: elongation of bonds in parallel, the zipper mode of unfolding, and elongation and unfolding of bonds in series. The two first elongation regions are dominated by a cooperative bond opening, in which each bond is influenced by its neighbor, whereas the third region can be described by individual bond opening, in which the bonds open and close randomly. A methodology for a swift extraction of model parameters from force-versus-elongation measurements performed under equilibrium conditions is derived. Entities such as the free energy, the stiffness, the elastic elongation, the opening length of the various bonds, and the number of PapA units in the rod are determined.
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| 11. |
- Andersson, Magnus, et al.
(författare)
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Dynamic Force Spectroscopy of E. coli P Pili
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 91:7, s. 2717-2725
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Tidskriftsartikel (refereegranskat)abstract
- Surface organelles (so-called pili) expressed on the bacterial membrane mediate the adhesion of Escherichia coli causing urinary tract infection. These pili possess some extraordinary elongation properties that are assumed to allow a close bacterium-to-host contact even in the presence of shear forces caused by urine flow. The elongation properties of P pili have therefore been assessed for low elongation speeds (steady-state conditions). This work reports on the behavior of P pili probed by dynamic force spectroscopy. A kinetic model for the unfolding of a helixlike chain structure is derived and verified. It is shown that the unfolding of the quaternary structure of the PapA rod takes place at a constant force that is almost independent of elongation speed for slow elongations (up to ~0.4 μm/s), whereas it shows a dynamic response with a logarithmic dependence for fast elongations. The results provide information about the energy landscape and reaction rates. The bond length and thermal bond opening and closure rates for the layer-to-layer bond have been assessed to ~0.76 nm, ~0.8 Hz, and ~8 GHz, respectively. The results also support a previously constructed sticky-chain model for elongation of the PapA rod that until now had been experimentally verified only under steady-state conditions.
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| 12. |
- Andersson, Magnus, et al.
(författare)
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The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment
- 2007
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 93:9, s. 3008-3014
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Tidskriftsartikel (refereegranskat)abstract
- Uropathogenic Escherichia coli express pili that mediate binding to host tissue cells. We demonstrate with in situ force measuring optical tweezers that the ability of P and type 1 pili to elongate by unfolding under exposure to stress is a shared property with some differences. The unfolding force of the quaternary structures under equilibrium conditions is similar, 28 ± 2 and 30 ± 2 pN for P pili and type 1 pili, respectively. However, type 1 pili are found to be more rigid than P pili through their stronger layer-to-layer bonds. It was found that type 1 pili enter a dynamic regime at elongation speeds of 6 nm/s, compared to 400 nm/s for P pili; i.e., it responds faster to an external force. This possibly helps type 1 to withstand the irregular urine flow in the urethra as compared to the more constant urine flow in the upper urinary tract. Also, it was found that type 1 pili refold during retraction at two different levels that possibly could be related to several possible configurations. Our findings highlight functions that are believed to be of importance for the bacterial ability to sustain a basic antimicrobial mechanism of the host and for bacterial colonization.
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| 13. |
- André, Ingemar, et al.
(författare)
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Salt enhances calmodulin-target interaction
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 90:8, s. 2903-2910
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin (CaM) operates as a Ca2+ sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca2+-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.
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| 14. |
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| 15. |
- Balaz, Martina, et al.
(författare)
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Protein-surface Interactions and Functional Geometry of Surface-adsorbed Myosin Motor Fragments
- 2009
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 96:3 Suppl. 1, s. 495A-495A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biophysical studies with myosin motor fragments (heavy meromyosin; HMM and subfragment 1; S1) adsorbed to artificial surfaces, are important for elucidation of actomyosin function. In spite of the widespread use of such in vitro motility assays and single molecule studies, little is known about the adsorption geometry and effects of protein-surface interactions on the motor properties. Here, we investigate these factors with focus on HMM using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) spectroscopy based ATPase assays. In the latter, we monitored the turnover of Alexa-fluor647-ATP (Alexa-ATP) by surface adsorbed HMM. Studies were performed with HMM/S1 adsorbed to model hydrophilic (SiO2) or hydrophobic (trimethyl-chlorosilane [TMCS] - derivatized) surfaces. The results suggest that adsorption of HMM is weakened on SiO2 (but not on TMCS) at high (245 mM) compared to low (65 mM) ionic strengths. The changes in ionic strength were also associated with structural changes in the protein layer according to QCM-D studies. Moreover, the TIRF based ATPase assay suggested a larger fraction of HMM molecules with low catalytic activity on SiO2. These and other TIRF and QCM-D results, suggest that HMM preferentially adsorbs to negatively charged hydrophilic surfaces via the actin-binding region. In contrast, the majority of the HMM molecules seem to adsorb via their C-terminal tail on moderately hydrophobic surfaces. In the latter case the catalytic sites appear to be close to, but not immobilized on the surface. The results with HMM were compared to, and found consistent with, QCM-D and TIRF-data obtained with S1 motor fragments.
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| 16. |
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| 17. |
- Balogh, Johanna, et al.
(författare)
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Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibres.
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 88:2, s. 1156-1165
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Tidskriftsartikel (refereegranskat)abstract
- Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des–/–) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des–/– muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des–/– and Des+/+. In width measurements of single fibers and bundles, Des–/– soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des–/– and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des–/– soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.
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| 18. |
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| 19. |
- Bárány-Wallje, Elsa, et al.
(författare)
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A critical reassessment of penetratin translocation across lipid membranes.
- 2005
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Ingår i: Biophys J. - 0006-3495. ; 89:4, s. 2513-21
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Tidskriftsartikel (refereegranskat)abstract
- Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophiliccargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference.A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is,<10^-13m/s.Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasmamembrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.
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| 20. |
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| 21. |
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| 22. |
- Benninger, Richard K. P., et al.
(författare)
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Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:2, s. L13-L15
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Tidskriftsartikel (refereegranskat)abstract
- We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.
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| 23. |
- Berntsen, Peter, 1974, et al.
(författare)
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Dielectric and calorimetric studies of hydrated purple membrane
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 89:5, s. 3111-3128
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Tidskriftsartikel (refereegranskat)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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| 24. |
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| 25. |
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| 26. |
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| 27. |
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| 28. |
- Borejdo, J., et al.
(författare)
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Application of surface plasmon coupled emission to study of muscle
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 91:7, s. 2626-2635
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Tidskriftsartikel (refereegranskat)abstract
- Muscle contraction results from interactions between actin and myosin cross-bridges. Dynamics of this interaction may be quite different in contracting muscle than in vitro because of the molecular crowding. In addition, each cross-bridge of contracting muscle is in a different stage of its mechanochemical cycle, and so temporal measurements are time averages. To avoid complications related to crowding and averaging, it is necessary to follow time behavior of a single cross-bridge in muscle. To be able to do so, it is necessary to collect data from an extremely small volume (an attoliter, 10 -18 liter). We report here on a novel microscopic application of surface plasmon-coupled emission (SPCE), which provides such a volume in a live sample. Muscle is fluorescently labeled and placed on a coverslip coated with a thin layer of noble metal. The laser beam is incident at a surface plasmon resonance (SPR) angle, at which it penetrates the metal layer and illuminates muscle by evanescent wave. The volume from which fluorescence emanates is a product of two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The fluorescence is quenched at the metal interface (up to ∼10 nm), which further limits the thickness of the fluorescent volume to ∼50 nm. The fluorescence is detected through a confocal aperture, which limits the lateral dimensions of the detection volume to ∼200 nm. The resulting volume is ∼2 × 10-18 liter. The method is particularly sensitive to rotational motions because of the strong dependence of the plasmon coupling on the orientation of excited transition dipole. We show that by using a high-numerical-aperture objective (1.65) and high-refractive-index coverslips coated with gold, it is possible to follow rotational motion of 12 actin molecules in muscle with millisecond time resolution. © 2006 by the Biophysical Society.
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| 29. |
- Brandt, Erik G., et al.
(författare)
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Dynamic structure factors from lipid membrane molecular dynamics simulations
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:5, s. 1828-1838
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic structure factors for a lipid bilayer have been calculated from molecular dynamics simulations. From trajectories of a system containing 1024 lipids we obtain wave vectors down to 0.34 nm(-1), which enables us to directly resolve the Rayleigh and Brillouin lines of the spectrum. The results confirm the validity of a model based on generalized hydrodynamics, but also improves the line widths and the position of the Brillouin lines. The improved resolution shows that the Rayleigh line is narrower than in earlier studies, which corresponds to a smaller thermal diffusivity. From a detailed analysis of the power spectrum, we can, in fact, distinguish two dispersive contributions to the elastic scattering. These translate to two exponential relaxation processes in separate time domains. Further, by including a first correction to the wave-vector-dependent position of the Brillouin lines, the results agree favorably to generalized hydrodynamics even up to intermediate wave vectors, and also yields a 20% higher adiabatic sound velocity. The width of the Brillouin lines shows a linear, not quadratic, dependence to low wave vectors.
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| 30. |
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| 31. |
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| 32. |
- Broomand, Amir, et al.
(författare)
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Electrostatic domino effect in the Shaker K channel turret
- 2007
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 93:7, s. 2307-2314
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated K channels are regulated by extracellular divalent cations such as Mg2+ and Sr2+, either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl2 shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at −20 mV and Kv2.1 channel open at +5 mV. The aim of this study was to identify critical residues for this Mg2+-induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg2+-shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425 > 419 > 451. Charging 425 and 419 not only affect the Mg2+-induced G(V) shift with 5–6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel’s working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4—thus, an electrostatic domino effect.
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| 33. |
- Börjesson, Sara, 1982-, et al.
(författare)
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Lipoelectric modification of ion channel voltage gating by polyunsaturated fatty acids
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 95:5, s. 2242-2253
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Tidskriftsartikel (refereegranskat)abstract
- Polyunsaturated fatty acids (PUFAs) have beneficial effects on epileptic seizures and cardiac arrhythmia. We report that ω-3 and ω-6 all-cis-PUFAs affected the voltage dependence of the Shaker K channel by shifting the conductance versus voltage and the gating charge versus voltage curves in negative direction along the voltage axis. Uncharged methyl esters of the PUFAs did not affect the voltage dependence, whereas changes of pH and charge mutations on the channel surface affected the size of the shifts. This suggests an electrostatic effect on the channel's voltage sensors. Monounsaturated and saturated fatty acids, as well as trans-PUFAs did not affect the voltage dependence. This suggests that fatty acid tails with two or more cis double bonds are required to place the negative carboxylate charge of the PUFA in a position to affect the channel's voltage dependence. We propose that charged lipophilic compounds could play a role in regulating neuronal excitability by electrostatically affecting the channel's voltage sensor. We believe this provides a new approach for pharmacological treatment that is voltage sensor pharmacology. © 2008 by the Biophysical Society.
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| 34. |
- Caballero-Herrera, A, et al.
(författare)
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Effect of urea on peptide conformation in water : Molecular dynamics and experimental characterization
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 89:2, s. 842-857
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations of a ribonuclease A C-peptide analog and a sequence variant were performed in water at 277 and 300 K and in 8 M urea to clarify the molecular denaturation mechanism induced by urea and the early events in protein unfolding. Spectroscopic characterization of the peptides showed that the C-peptide analog had a high alpha-helical content, which was not the case for the variant. In the simulations, interdependent side-chain interactions were responsible for the high stability of the alpha-helical C-peptide analog in the different solvents. The other peptide displayed alpha-helical unwinding that propagated cooperatively toward the N-terminal. The conformations sampled by the peptides depended on their sequence and on the solvent. The ability of water molecules to form hydrogen bonds to the peptide as well as the hydrogen bond lifetimes increased in the presence of urea, whereas water mobility was reduced near the peptide. Urea accumulated in excess around the peptide, to which it formed long-lived hydrogen bonds. The unfolding mechanisms induced by thermal denaturation and by urea are of a different nature, with urea-aqueous solutions providing a better peptide solvation than pure water. Our results suggest that the effect of urea on the chemical denaturation process involves both the direct and indirect mechanisms.
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| 35. |
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| 36. |
- de la Serna, Jorge Bernardino, et al.
(författare)
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Segregated Phases in Pulmonary Surfactant Membranes Do Not Show Coexistence of Lipid Populations with Differentiated Dynamic Properties
- 2009
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Ingår i: Biophysical Journal. - : Elsevier Inc. - 0006-3495. ; 97:5, s. 1381-1389
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Tidskriftsartikel (refereegranskat)abstract
- The composition of pulmonary surfactant membranes and films has evolved to support a complex lateral structure, including segregation of ordered/disordered phases maintained up to physiological temperatures. In this study, we have analyzed the temperature-dependent dynamic properties of native surfactant membranes and membranes reconstituted from two surfactant hydrophobic fractions (i.e., all the lipids plus the hydrophobic proteins SP-B and SP-C, or only the total lipid fraction). These preparations show micrometer-sized fluid ordered/disordered phase coexistence, associated with a broad endothermic transition ending close to 37°C. However, both types of membrane exhibit uniform lipid mobility when analyzed by electron paramagnetic resonance with different spin-labeled phospholipids. A similar feature is observed with pulse-field gradient NMR experiments on oriented membranes reconstituted from the two types of surfactant hydrophobic extract. These latter results suggest that lipid dynamics are similar in the coexisting fluid phases observed by fluorescence microscopy. Additionally, it is found that surfactant proteins significantly reduce the average intramolecular lipid mobility and translational diffusion of phospholipids in the membranes, and that removal of cholesterol has a profound impact on both the lateral structure and dynamics of surfactant lipid membranes. We believe that the particular lipid composition of surfactant imposes a highly dynamic framework on the membrane structure, as well as maintains a lateral organization that is poised at the edge of critical transitions occurring under physiological conditions.
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| 37. |
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| 38. |
- Deleu, Magali, et al.
(författare)
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Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes
- 2008
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 94:7, s. 2667-2679
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Tidskriftsartikel (refereegranskat)abstract
- Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.
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| 39. |
- Dell'Orco, D, et al.
(författare)
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Electrostatic contributions to the kinetics and thermodynamics of protein assembly
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 88:3, s. 1991-2002
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Tidskriftsartikel (refereegranskat)abstract
- The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D-9k reconstitution was measured in the presence of Ca2+ using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K-A = 1.3 x 10(10) M-1; K-D = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.
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| 40. |
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| 41. |
- Dias, Rita, et al.
(författare)
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Colloid Adsorption onto Responsive Membranes.
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 94:10, s. 3760-3768
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Tidskriftsartikel (refereegranskat)abstract
- The adsorption of colloids with varying sizes and charges onto a surface carrying both negative and positive charges representing a membrane has been investigated by using a simple model employing Monte Carlo simulations. The membrane is made of positive and negative charges (headgroups) that are allowed to move along the membrane, simulating the translational diffusion of the lipids, and are also allow to protrude into the solution, giving rise to a fluid and soft membrane. When an uncharged colloid is placed in the vicinity of the membrane, a short-range repulsion between the colloid and the membrane is observed and the membrane will deflect to avoid the contact with the colloid. When the colloid is charged, the membrane response is two-fold: the headgroups of the membrane move towards the colloid as to partly embrace it, and the positive headgroups of the membrane approach the oppositely charged colloid, inducing the demixing of the lipids (polarization) of the membrane. The presence of protrusions enhances the polarization of the membrane. Potential of mean force calculations show that protrusions give rise to a more long-ranged attractive colloid-membrane potential which however has a smaller magnitude at short separations.
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| 42. |
- Dinic, Jelena, et al.
(författare)
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Plasma membrane order in T cell signalling
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Plasma membrane nanodomains, referred to as lipid rafts, more ordered than the bulk membrane play an important role in T cell signalling by forming signalling platforms in activated T cells. However, the existence of lipid rafts in resting T cells is contentious. Using laurdan, a membrane probe whose peak emission wavelength depends on the lipid environment, evidence is presented for the existence of ordered nanodomains in resting T cells. T cell signalling can be initiated by stimulating the T cell receptor (TCR), crosslinking the lipid raft markers GM1 (sphingolipid) or glycosylphosphatidylinositol (GPI) anchored proteins. The aggregation of lipid raft components induces the same response in Jurkat T cells as the ligation of an antigen to the TCR. Changes in membrane order linked with reorganization of the plasma membrane upon Jurkat T cell activation were followed at 37°C. Fluorescent images were analyzed for generalised polarisation values - a measure of the relative abundance of liquid ordered and liquid disordered domains. TCR patching does not increase the overall membrane order suggesting that membrane domains of high order are brought together in the patches. This supports the existence of small ordered membrane domains in resting T cells that aggregate upon activation. Patching of GM1, the GPI-anchored protein CD59 and the non lipid raft marker CD45 significantly increases the overall membrane order. So does general crosslinking of membrane components with Concanavalin A. Remodelling of the actin cytoskeleton is an integral part of TCR signaling and T cell activation. Disrupting actin polymerization using latrunculin B decreases membrane order and stabilizing actin filaments with jasplakinolide increases membrane order. An increase in membrane order appears to be a general effect of plasma membrane component patching and is likely due to a global induction of actin polymerization at the plasma membrane.
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| 43. |
- Edholm, Olle, et al.
(författare)
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Areas of molecules in membranes consisting of mixtures
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 89:3, s. 1827-1832
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Tidskriftsartikel (refereegranskat)abstract
- The question has arisen in recent literature: how to partition the total area in simulations of membranes consisting of more than one kind of molecule into average areas for each kind of molecule. Several definitions have been proposed, each of which has arbitrary features. When applied to mixtures of cholesterol and DPPC, these definitions give different results. This note recalls that physical chemistry provides a canonical way to de. ne molecular area, in analogy to the definition of partial-specific volume. Results for partial-specific area are obtained from simulations of DPPC/cholesterol bilayers and compared to the results from the other recent definitions. The partial-specific-area formalism dramatically demonstrates the condensing effect of cholesterol and this leads to the introduction of a specific model that accounts for the area of mixtures of cholesterol and lipid over the entire range of cholesterol concentrations.
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| 44. |
- Edholm, O
(författare)
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Dynamics of lipid bilayers from molecular dynamics simulations
- 2005
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Ingår i: Biophysical Journal. - Royal Inst Technol, Dept Phys, AlbaNova Univ Ctr, SE-10691 Stockholm, Sweden. : BIOPHYSICAL SOCIETY. - 0006-3495 .- 1542-0086. ; 88:1, s. 26A-26A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 45. |
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| 46. |
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| 47. |
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| 48. |
- Evilevitch, Alex, et al.
(författare)
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Effects of salt concentrations and bending energy on the extent of ejection of phage genomes
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 94:3, s. 1110-1120
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Tidskriftsartikel (refereegranskat)abstract
- Recent work has shown that pressures inside dsDNA phage capsids can be as high as many tens of atmospheres; it is this pressure that is responsible for initiation of the delivery of phage genomes to host cells. The forces driving ejection of the genome have been shown to decrease monotonically as ejection proceeds, and hence to be strongly dependent on the genome length. Here we investigate the effects of ambient salts on the pressures inside phage-l, for the cases of mono-, di-, and tetravalent cations, and measure how the extent of ejection against a fixed osmotic pressure (mimicking the bacterial cytoplasm) varies with cation concentration. We find, for example, that the ejection fraction is halved in 30 mM Mg21 and is decreased by a factor of 10 upon addition of 1 mM spermine. These effects are calculated from a simple model of genome packaging, using DNA-DNA repulsion energies as determined independently from x-ray diffraction measurements on bulk DNA solutions. By comparing the measured ejection fractions with values implied from the bulk DNA solution data, we predict that the bending energy makes the d- spacings inside the capsid larger than those for bulk DNA at the same osmotic pressure.
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| 49. |
- Falck, Emma, et al.
(författare)
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Interaction of fusidic acid with lipid membranes: Implications to the mechanism of antibiotic activity
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 91:5, s. 1787-1799
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the effects of cholesterol and steroid-based antibiotic fusidic acid (FA) on the behavior of lipid bilayers using a variety of experimental techniques together with atomic-scale molecular dynamics simulations. Capillary electrophoretic measurements showed that FA was incorporated into fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes. Differential scanning calorimetry in turn showed that FA only slightly altered the thermodynamic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, whereas cholesterol abolished all endotherms when the mole fraction of cholesterol (X-chol) was > 0.20. Fluorescence spectroscopy was then used to further characterize the influence of these two steroids on DPPC large unilamellar vesicles. In the case of FA, our result strongly suggested that FA was organized into lateral microdomains with increased water penetration into the membrane. For cholesterol/DPPC mixtures, fluorescence spectroscopy results were compatible with the formation of the liquid-ordered phase. A comparison of FA and cholesterol-induced effects on DPPC bilayers through atomistic molecular dynamics simulations showed that both FA and cholesterol tend to order neighboring lipid chains. However, the ordering effect of FA was slightly weaker than that of cholesterol, and especially for deprotonated FA the difference was significant. Summarizing, our results show that FA is readily incorporated into the lipid bilayer where it is likely to be enriched into lateral microdomains. These domains could facilitate the association of elongation actor-G into lipid rafts in living bacteria, enhancing markedly the antibiotic efficacy of FA.
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| 50. |
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