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Träfflista för sökning "L773:0006 3495 OR L773:1542 0086 srt2:(2005-2009)"

Sökning: L773:0006 3495 OR L773:1542 0086 > (2005-2009)

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1.
  • Benninger, R. K. P., et al. (författare)
  • Fluorescence imaging of two-photon linear dichroism : Cholesterol depletion disrupts molecular orientation in cell membranes
  • 2005
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:1, s. 609-622
  • 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)
  • Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy
  • 2005
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:6, s. 4200-4212
  • 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)
  • Plasma membrane topology and membrane models
  • 2009
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 3:Supplement 1, s. 282a-282a
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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4.
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5.
  • Akanda, Nesar, et al. (författare)
  • Biophysical properties of the apoptosis-inducing plasma membrane voltage-dependent anion channel
  • 2006
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 90:12, s. 4405-4417
  • 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.
  • Andér, Martin, 1979-, et al. (författare)
  • Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
  • 2008
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 94:3, s. 820-831
  • 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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7.
  • Andersson, Magnus, et al. (författare)
  • A sticky chain model of the elongation and unfolding of escherichia coli P pili under stress
  • 2006
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 90:5, s. 1521-1534
  • 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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8.
  • Andersson, Magnus, et al. (författare)
  • Dynamic Force Spectroscopy of E. coli P Pili
  • 2006
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 91:7, s. 2717-2725
  • 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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9.
  • Andersson, Magnus, et al. (författare)
  • The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment
  • 2007
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 93:9, s. 3008-3014
  • 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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10.
  • André, Ingemar, et al. (författare)
  • Salt enhances calmodulin-target interaction
  • 2006
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 90:8, s. 2903-2910
  • 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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