European Biophysics Journal (EUR BIOPHYS J BIOPHY)

Publications in this journal

• Article: Folding dynamics of phenylalanine hydroxylase depends on the enzyme’s metallation state: the native metal, iron, protects against aggregate intermediates

  Aristobulo Loaiza, Judith A. Ronau, Alexander Ribbe, Lia Stanciu, John W. Burgner, Lake N. Paul, Mahdi M. Abu-Omar
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  ABSTRACT: Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for the phenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU). To better understand how protein structure and folding profiles are affected by the metal cofactor, we investigated the chemical (un)folding of apo- and holo-PAH from Chromobacterium violaceum (cPAH) using circular dichroism (CD) and analytical ultracentrifugation (AUC). Holo-cPAH shows a two-state unfolding transition. In contrast, the unfolding profile for apo-cPAH reveals a three-state (un)folding pathway and accumulation of an intermediate (apo-cPAHI). This intermediate is also observed in refolding experiments. Fluorescence studies are consistent with the CD findings. The intermediate apo-cPAHI and unfolded state(s) of apo- and holo-cPAHU have been characterized by analytical ultracentrifugation (AUC). At 2.4 and 2.8M GuHCl, 90% of the signal for apo-cPAH has a weight average sedimentation coefficient in water at 20°C (s20,w) of about 48S, representing multiple aggregate species made of multiple monomers of cPAH. Aggregate formation for apo-cPAH is also confirmed by dynamic light scattering and electron microscopy giving a hydrodynamic radius (RH) of 41nm for apo-cPAHI versus 3.5nm for the native protein. KeywordsProtein folding–Thermodynamics–Circular dichroism–Fluorescence–Metalloprotein–Analytical ultracentrifugation
  European Biophysics Journal 04/2012; 40(8):959-968.
• Article: Guanidine hydrochloride and urea-induced unfolding of Brugia malayi hexokinase

  Alok Ranjan Singh, Shweta Joshi, Rahul Arya, Arvind Mohan Kayastha, Jitendra Kumar Saxena
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  ABSTRACT: Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl) and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured by use of high concentrations of GdmCl or urea.
  European Biophysics Journal 04/2012; 39(2):289-297.
• Article: An alternative flexible conformation of the E. coli HUβ2 protein: structural, dynamics, and functional aspects

  Norbert Garnier, Karine Loth, Franck Coste, Rafal Augustyniak, Virginie Nadan, Christian Damblon, Bertrand Castaing
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  ABSTRACT: The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα2, EcHUβ2, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N2↔I2) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N2 to I2 thermal transition of the EcHUβ2 homodimer. On the basis of these data, a realistic 3D model is proposed for the major I2 conformation of EcHUβ2. This model is in agreement with previous experimental data. KeywordsHistone-like HU protein–Thermal stability–Structure flexibility–Molecular dynamics simulation–NMR
  European Biophysics Journal 04/2012; 40(2):117-129.
• Article: Highly polar environments catalyze the unfolding of PrPC helix 1

  Martin Lingenheil, Robert Denschlag, Paul Tavan
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  ABSTRACT: The first α-helix (H1) likely plays an important role in the conversion of the cellular prion protein (PrPC) into its pathogenic isoform (PrPSc). In this conversion, H1 may either have to unfold or may represent a site of intermolecular contact. A recent molecular dynamics simulation suggested that H1 can unfold if it is detached from the protein core (Hirschberger etal. in Biophys J 90:3908, 2006). It has been hypothesized that the high dielectric constant ε S of the bulk water environment facilitates the unfolding of H1. To check this hypothesis, we performed a number of replica exchange molecular dynamics simulations of an H1 peptide in solvents of different ε S . We found that the equilibrium helix fraction in water is less than 40%, in agreement with previous experimental findings, and that the helix unfolds much faster in water than in less polar solvents. The kinetically stabilizing effect of the organic solvents is largely unspecific and correlates well with their dielectric constant ε S . KeywordsMolecular dynamics-Peptide studies-Secondary structure-Prion protein-Helix 1-Dielectric constant
  European Biophysics Journal 04/2012; 39(8):1177-1192.
• Article: Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents

  Vladimir N. Vorob’ev, Alexander V. Anisimov, Nailya R. Dautova
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  ABSTRACT: The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05mM, and reaching a plateau at a concentration of 0.5mM.
  European Biophysics Journal 04/2012; 37(6):975-978.
• Article: Formation of a molten globule like state in bovine serum albumin at alkaline pH

  Priyankar Sen, Basir Ahmad, Rizwan Hasan Khan
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  ABSTRACT: Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.
  European Biophysics Journal 04/2012; 37(8):1303-1308.
• Article: Metal effects on the membrane interactions of amyloid-β peptides

  John D. Gehman, Caitlin C. O’Brien, Fazel Shabanpoor, John D. Wade, Frances Separovic
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  ABSTRACT: Aβ(1–42) peptide, found as aggregated species in Alzheimer’s disease brain, is linked to the onset of dementia. We detail results of 31P and 2H solid-state NMR studies of model membranes with Aβ peptides and the effect of metal ions (Cu2+ and Zn2+), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when Aβ(1–42) is associated with the bilayer membrane. Aβ(25–35), a fragment from the C-terminal end of Aβ(1–42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with Aβ(25–35) peptides. 31P magic angle spinning NMR data show that Aβ(1–42) and Aβ(1–42)-Cu2+ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state 13C NMR was used to compare structural changes of Aβ(1–42) to those of Aβ(25–35) in model membrane systems of anionic phospholipids and cholesterol. The Aβ peptides appeared to have an increase in β-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu2+ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated Aβ/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide.
  European Biophysics Journal 04/2012; 37(3):333-344.
• Article: Functional imaging of mucociliary phenomena

  M. Ryser, A. Burn, Th. Wessel, M. Frenz, J. Rička
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  ABSTRACT: We present a technique for the investigation of mucociliary phenomena on trachea explants under conditions resembling those in the respiratory tract. Using an enhanced reflection contrast, we detect simultaneously the wave-like modulation of the mucus surface by the underlying ciliary activity and the transport of particles embedded in the mucus layer. Digital recordings taken at a speed of 500 frames per second are analyzed by a set of refined data processing algorithms. The simultaneously extracted data include not only ciliary beat frequency and its surface distribution, but also space–time structure of the mucociliary wave field, wave velocity and mucus transport velocity. Furthermore, we propose the analysis of the space and time evolution of the phase of the mucociliary oscillations to be the most direct way to visualize the coordination of the cilia. In particular, this analysis indicates that the synchronization is restricted to patches with varying directions of wave propagation, but the transport direction is strongly correlated with the mean direction of waves. The capabilities of the technique and of the data-processing algorithms are documented by characteristic data obtained from mammalian and avine tracheae.
  European Biophysics Journal 04/2012; 37(1):35-54.
• Article: Ligand-escape pathways from the ligand-binding domain of PPARγ receptor as probed by molecular dynamics simulations

  D. Genest, N. Garnier, A. Arrault, C. Marot, L. Morin-Allory, M. Genest
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  ABSTRACT: Conformational rearrangements of peroxysome proliferator activated receptor (PPARγ) ligand-binding domain (LBD) that accompany the release and binding of ligands are not well understood. To determine the major events associated with the escape of the partial agonist GW0072, molecular dynamic (MD) simulations were performed using two different methods: reversed targeted molecular dynamics (TMD−1) and time-dependent distance restraints (TDR) using as restraints either the root mean square deviation from a reference structure (TMD−1) or the distance between the geometrical centers of the binding pocket and of the ligand (TDR). Both methods do not assume any a priori route for ligand extraction. To avoid artifacts, different initial simulation conditions were used and particular attention was paid for giving time to the protein to relax during the extraction process by running 10–12ns simulations within explicit water. Two distinct exit gates A and B were found, independently of initial conditions and method. During the exit process no interaction between GW0072 and the transactivation AF-2 helix was observed. Our results suggest that the ligand uses the intrinsic flexibility of the protein to move within the receptor. Paths A and B are very similar to those found for other nuclear receptors, suggesting that these routes are a common characteristics of nuclear receptors that are used by different kinds of ligands. Finally, the knowledge of entry/exit pathways of a receptor should be very useful in discriminating between different ligands that could have been favorably docked in the binding pocket by introducing docking along these pathways into computational drug design protocols.
  European Biophysics Journal 04/2012; 37(4):369-379.
• Article: Cholesterol inhibits the insertion of the Alzheimer’s peptide Aβ(25–35) in lipid bilayers

  Silvia Dante, Thomas Hauß, Norbert A. Dencher
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  ABSTRACT: The physiological relationship between brain cholesterol content and the action of amyloid β (Aβ) peptide in Alzheimer’s disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Aβ/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Aβ(25–35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Aβ(25–35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Aβ(25–35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Aβ(25–35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties.
  European Biophysics Journal 04/2012; 35(6):523-531.
• Article: 39K nuclear magnetic resonance and a mathematical model of K+ transport in human erythrocytes

  Anthony D. Maher, Bogdan E. Chapman, Philip W. Kuchel
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  ABSTRACT: 39K nuclear magnetic resonance was used to measure the efflux of K+ from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of 39K+ were selected on the basis of their longitudinal relaxation times, T 1, by using an inversion- recovery pulse sequence with the mixing time, τ1, chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T 1 values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K+ efflux mediated by the Gárdos channel was 1.19±0.40mmol (L RBC)−1min−1 at 37°C.
  European Biophysics Journal 04/2012; 35(4):293-301.
• Article: Lipid membrane-induced optimization for ligand–receptor docking: recent tools and insights for the “membrane catalysis” model

  Miguel A.R.B. Castanho, Miguel X. Fernandes
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  ABSTRACT: Cells in living organisms are regulated by chemical and physical stimuli from their environment. Often, ligands interact with membrane receptors to trigger responses and Sargent and Schwyzer conceived a model to describe this process, “membrane catalysis”. There is a notion that the physical organization of membranes can control the response of cells by speeding up reactions. We revisit the “membrane catalysis” model in the light of recent technical, methodological and theoretical advances and how they can be exploited to highlight the details of membrane mediated ligand–receptor interactions. We examine the possible effects that ligand concentration causes in the membrane catalysis and focus our attention in techniques used to determine the partition constant. The hypothetical diffusional advantage associated with membrane catalysis is discussed and the applicability of existing models is assessed. The role of in-depth location and orientation of ligands is explored emphasizing the contribution of new analysis methods and spectroscopic techniques. Results suggest that membranes can optimize the interaction between ligands and receptors through several different effects but the relative contribution of each must be carefully investigated. We certainly hope that the conjugation of the methodological and technical advances here reported will revive the interest in the membrane catalysis model.
  European Biophysics Journal 04/2012; 35(2):92-103.
• Article: A nonlinear model of ionic wave propagation along microtubules

  M. V. Satarić, D. I. Ilić, N. Ralević, Jack Adam Tuszynski
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  ABSTRACT: Microtubules (MTs) are important cytoskeletal polymers engaged in a number of specific cellular activities including the traffic of organelles using motor proteins, cellular architecture and motility, cell division and a possible participation in information processing within neuronal functioning. How MTs operate and process electrical information is still largely unknown. In this paper we investigate the conditions enabling MTs to act as electrical transmission lines for ion flows along their lengths. We introduce a model in which each tubulin dimer is viewed as an electric element with a capacitive, inductive and resistive characteristics arising due to polyelectrolyte nature of MTs. Based on Kirchhoff’s laws taken in the continuum limit, a nonlinear partial differential equation is derived and analyzed. We demonstrate that it can be used to describe the electrostatic potential coupled to the propagating localized ionic waves.
  European Biophysics Journal 04/2012; 38(5):637-647.
• Article: Human erythrocyte flickering: temperature, ATP concentration, water transport, and cell aging, plus a computer simulation

  David Szekely, Tsz Wai Yau, Philip W. Kuchel
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  ABSTRACT: Images of human erythrocytes from a healthy donor were recorded under differential interference contrast (DIC) microscopy; they were acquired rapidly (~336Hz) and the intensity of the centermost pixel of each cell was recorded for ~60s (20,000 values). Various techniques were used to analyze the data, including detrended fluctuation analysis (DFA) and multiscale entropy (MSE); however, power spectrum analysis was deemed the most appropriate for metrifying and comparing results. This analysis was used to compare cells from young and old populations, and after perturbing normal conditions, with changes in temperature, adenosine triphosphate (ATP) concentration (using NaF, an inhibitor of glycolysis, and α-toxin, a pore-forming molecule used to permeabilize red cells to ATP), and water transport rates [using glycerol, and p-chloromercuriphenylsulfonic acid (pCMBS) to inhibit aquaporins, AQPs]. There were measurable differences in the membrane fluctuation characteristics in populations of young and old cells, but there was no significant change in the flickering time series on changing the temperature of an individual cell, by depleting it of ATP, or by competing with the minor water exchange pathway via AQP3 using glycerol. However, pCMBS, which inhibits AQP1, the major water exchange pathway, inhibited flickering in all cells, and yet it was restored by the membrane intercalating species dibutyl phthalate (DBP). We developed a computer model to simulate acquired displacement spectral time courses and to evaluate various methods of data analysis, and showed how the flexibility of the membrane, as defined in the model, affects the flickering time course.
  European Biophysics Journal 04/2012; 38(7):923-939.
• Article: Biographical memoir: Alexander Beaumont Hope, Australian biophysicist, 1928–2008

  Peter H. Barry, Hans G. L. Coster, Wah Soon Chow
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  ABSTRACT: This introductory article is the first of four short articles from the Tribute to Alex Hope Symposium held at the 2008 Australian Society for Biophysics meeting in Canberra, Australia, as a tribute to Professor Alex Hope, who died in July last year. As well as briefly introducing the other three articles by three former PhD students, it will also be a biographical memoir of Alex Hope.
  European Biophysics Journal 04/2012; 39(1):175-178.
• Article: Luminal Ca2+ activation of cardiac ryanodine receptors by luminal and cytoplasmic domains

  Derek R. Laver
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  ABSTRACT: The ryanodine receptors form the calcium release channel in the membrane of the sarcoplasmic reticulum (SR, the main intracellular Ca2+ store). The importance of ryanodine receptors (RyRs) to cardiac pacemaking and rhythmicity is highlighted by more than 69 mutations, RyR mutations, which underlie arrhythmias and sudden cardiac death. Although most of these mutations lie in cytoplasmic domains, they all cause increased RyR activation by Ca2+ in the SR lumen. Presented here is a review of the mechanisms by which cytoplasmic domains of the RyR can determine luminal activation.
  European Biophysics Journal 04/2012; 39(1):19-26.
• Article: The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

  Shimshon Ben Dror, Irena Bronshtein, Hana Weitman, Kevin M. Smith, William G. O’Neal, Peter A. Jacobi, Benjamin Ehrenberg
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  ABSTRACT: In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed.
  European Biophysics Journal 04/2012; 38(7):847-855.