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Biophysical Chemistry - Verlag: Elsevier
'Biophysical Chemistry' publiziert Originalarbeiten und Bewertungen in den Bereichen Chemie und Physik mit direkten Einfluss auf biologischer Phänomene. Die quantitative Analyse der Eigenschaften von biologischen Makromolekülen, biologisch aktive Moleküle, makromolekulare Gruppen und Zellbestandteile Kinetik, Thermodynamik, raum-zeitliche Organisation, NMR- und Röntgen-Strukturbiologie, sowie Einzel-Molekül-Erkennung sind wichtige Schwerpunkte der Zeitschrift.
Publication year: 2012 Source: Biophysical Chemistry, Available online 8 January 2012 Yipin Zhou, Pedro Cabrales, Andre F. Palmer A mathematical model was developed to study nitric oxide (NO) and oxygen (O2) transport in an arteriole and surrounding tissues exposed to a mixture of red blood cells (RBCs) and hemoglobin (Hb)-based O2carriers (HBOCs). A unique feature of this model is the inclusion of blood vessel wall shear stress-induced production of endothelial-derived NO, which is very sensitive to the viscosity of the RBC and HBOC mixture traversing the blood vessel lumen. Therefore in this study, a series of polymerized bovine Hb (PolyHb) solutions with high viscosity, varying O2affinity, NO dioxygenation rate constants and O2dissociation rate constants that were previously synthesized and characterized by our group were evaluated via mathematical modeling, in order to investigate the effect of these biophysical properties on the transport of NO and O2in an arteriole and its surrounding tissues subjected to anemia with the commercial HBOC Oxyglobin® and cell-free bovine Hb (bHb) serving as appropriate controls. The computer simulation results indicated that transfusion of high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO, especially in the blood vessel wall and should transport enough NO inside the smooth muscle layer to activate vasodilation compared to the commercial HBOC Oxyglobin® and cell-free bHb. However, NO scavenging in the arteriole lumen was unavoidable due to the intrinsic high NO dioxygenation rate constant of the HBOCs being studied. This study also observed that all PolyHbs could potentially improve tissue oxygenation under hypoxic conditions, while low O2affinity PolyHbs were more effective in oxygenating tissues under normoxic conditions compared with high O2affinity PolyHbs. In addition, all ultrahigh molecular weight PolyHbs displayed higher O2transfer rates than the commercial HBOC Oxyglobin® and cell-free bHb. Therefore, these results suggest that ultrahigh molecular weight PolyHb solutions could be used as safe and efficacious O2carriers for use in transfusion medicine. It also suggests that future generations of PolyHb solutions should possess lower NO dioxygenation reaction rate constants in order to reduce NO scavenging, while maintaining high solution viscosity to take advantage of wall shear stress-induced NO production. Taken together, we suggest that this mathematical model can be used to predict the vasoactivity of HBOCs and help guide the design and optimization of the next generation of HBOCs for use in transfusion medicine.
Highlights
? Developed mathematical model of nitric oxide and oxygen transport in an arteriole. ? Arteriole contained a mixture of red blood cells and HBOCs. ? Model accounted for wall shear stress-induced production of nitric oxide.
Publication year: 2012 Source: Biophysical Chemistry, Available online 5 January 2012 Katja Wegner, Anastasia Bachmann, Jan-Ulrich Schad, Philippe Lucarelli, Sven Sahle, ... Transforming growth factor?(TGF-?) ligands activate a signaling cascade with multiple cell context dependent outcomes. Disruption or disturbance leads to variant clinical disorders. To develop strategies for disease intervention, delineation of the pathway in further detail is required. Current theoretical models of this pathway describe production and degradation of signal mediating proteins and signal transduction from the cell surface into the nucleus, whereas feedback loops have not exhaustively been included.In this study we present a mathematical model to determine the relevance of feedback regulators (Arkadia, Smad7, Smurf1, Smurf2, SnoN and Ski) on TGF-?target gene expression and the potential to initiate stable oscillations within a realistic parameter space. We employed massive sampling of the parameters space to pinpoint crucial players for potential oscillations as well as transcriptional product levels. We identified Smad7 and Smurf2 with the highest impact on the dynamics. Based on these findings, we conducted preliminary time course experiments.
Highlights
? We present a mathematical model to determine the relevance of feedback loops in the TGF-? signaling. ? Our computational analysis indicated that oscillations are possible under realistic conditions. ? Smad7 and Smurf1/2 have the strongest potential to act as negative feedback regulators. ? Ski, SnoN, SARA, Arkadia have a high potential to also act as positive feedback mechanisms. ? Preliminary experimental data revealed a more dynamic behavior than the simple on-off mechanism.
Publication year: 2011 Source: Biophysical Chemistry, Available online 29 December 2011 Marie-Michèle Guy, Normand Voyer In order to study the impact of the amino acid sequence on the morphology of peptide-based nanostructures and their hydrogel formation, we designed a series of analogues of a milk-derived octapeptide (OP), mainly using strategic amino acid substitutions. Electronic transmission microscopy (TEM) and circular dichroism (CD) spectropolarimetry were used to analyze the nanostructures formed, and to characterize some structural features of the modified peptides. Further, the potential to form hydrogels was investigated for all of the analogous peptides. We learned that those able to undergo secondary structure transition to ?-sheet conformation form strong gels. The results reported highlight some key structural properties that explain the self-assembly propensity of Peptide OP.
Highlights
? Analogues of a milk-derived octapeptide are used to study the relationship between structure and gelation ability. ? ß-sheet conformation propensity correlates with the ability of the peptides to self-assemble into nanofibers and to form hydrogel. ? Key structural features important for peptide-based hydrogel formation are identified. ? Results open new avenues for molecular engineering of peptide based-hydrogels from edible sources.
Publication year: 2011 Source: Biophysical Chemistry, Available online 27 December 2011 Okimasa Okada, Kei Odai, Tohru Sugimoto, Etsuro Ito The gating of ion channel of ionotropic glutamate receptors is controlled by the structural change of the ligand-binding domain of GluR2. We examined the roles of residues in the glutamate-binding and cleft-closing mechanisms by molecular dynamics (MD) simulations. A glutamate entered the cleft deeply within the order of nanoseconds and the cleft locked the glutamate completely at 15 ns in an MD run. TYR450 seemed to regulate the orientation of the glutamate upon binding by cation-? interaction. A semi-open state was identified in the free energy profile evaluated with the structures on the spontaneously glutamate-bound and cleft-closed pathway by the unbiased MD simulations for the first time to our knowledge. In the semi-open state, the two sub-domains were bridged by two hydrogen bonds of GLU705 in the sub-domain 2 with TYR732 in the sub-domain 1 and with the glutamate bound to the sub-domain 1 until the transition to the closed state.
Highlights
? Unbiased MD simulations reveal the spontaneous glutamate-binding and cleft-closing mechanisms. ? The glutamate is attracted by ARG485 to enter the cleft from a more flexible side without the S-S bond. ? The orientation of the glutamate upon binding is regulated by TYR450 with the cation-? interaction. ? A semi-open state is identified in the free energy profile obtained by the unbiased MD simulations. ? The so-called linear response theory is verified in the inter-domain motion of the LBD of GluR2.
Publication year: 2011 Source: Biophysical Chemistry, Available online 24 December 2011 Mathieu Arseneault, Michel Dumont, François Otis, Normand Voyer We have prepared fluorescent analogs of known ion-channel-forming synthetic peptide nanostructures. These analogs were designed as probes to gain insight about the mechanism by which self-assembling amphiphilic peptides interact with lipid membranes. Conformational studies demonstrated that the labeled analogs retain their propensity to adopt a strong helical conformation in 2,2,2-trifluoroethanol and lipid bilayers. Attenuated total reflectance results indicated that the fluorescent peptide nanostructures are under an incorporation equilibrium between two forms, adsorbed at the surface or incorporated within the bilayer, similar to their unlabeled counterparts. However, when using a HeLa mimicking membrane, the proportion of peptide nanostructures in the transmembrane orientation decreases significantly. Finally, we were able to show by confocal microscopy studies that fluorescent analogs internalized into HeLa cells and localized into both the membranes of inner organelles and the cell membrane.
Highlights
? We prepared fluorescently labeled analogs of ion-channel-forming peptide nanostructures. ? The compounds retain their propensity to adopt a helical conformation within lipid membranes. ? Adding a fluorescent probe decreases the nanostructures' ability to insert in a transmembrane orientation. ? The peptide nanostructures enter living cells and localize into both organelle and cell membranes.
Publication year: 2011 Source: Biophysical Chemistry, Available online 24 December 2011 Anne Meunier, Rémy Fulcrand, François Darchen, Manon Guille Collignon, Frédéric Lemaître, ... The microfabrication and successful testing of a series of three ITO (Indium Tin Oxide) microsystems for amperometric detection of cells exocytosis are reported. These microdevices have been optimized in order to simultaneously (i) enhance signal to noise ratios, as required electrochemical monitoring, by defining appropriate electrodes geometry and size, and (ii) provide surface conditions which allow cells to be cultured over during one or two days, through apposite deposition of a collagen film. The intrinsic electrochemical quality of the microdevices as well as the effect of different collagen treatments were assessed by investigating the voltammetric responses of two classical redox systems, Ru(NH3)6and Fe(CN)6. This established that a moderate collagen treatment does not incur any significant alteration of voltammetric responses or degradation of the excellent signal-to-noise ratio.Among these three microdevices, the most versatile one involved a configuration in which the ITO microelectrodes were delimited by a microchannel coiled into a spiral. Though providing extremely good electrochemical responses this specific design allowed proper seeding and culture of cells permitting either single cell or cell cluster stimulation and analysis.
Highlights
? Microfabrication of three microsystems for amperometric detection of exocytosis. ? Effect of collagen treatments on the electrochemical capability. ? Moderate collagen treatment does not induce any alteration of voltammetric responses or degradation of the signal-to-noise ratio.
Publication year: 2011 Source: Biophysical Chemistry, Available online 7 December 2011 Shigefumi Mimura, Takahisa Yamato, Tadashi Kamiyama, Kunihiko Gekko The evolution of structural fluctuations of proteins was examined by calculating the isothermal compressibility (?T) values of chicken lysozyme and its six evolutionary mutants at Thr40, Ile55, and Ser91 (a ternary mutant corresponding to bobwhite lysozyme) from their X-ray structures by normal-mode analysis at 300 K. The ?Tvalues of the two extant lysozymes from chicken and bobwhite were 1.61 and 1.59 Mbar, respectively, but five other evolutionary mutants showed larger ?Tvalues of up to 2.17 Mbar. These results suggest that ancestral lysozymes exhibit larger volume fluctuations than extant ones, and hence that the molecular evolution of lysozymes has followed a nonneutral evolutionary pathway. The evolutionary mutants contained large amount of cavities, although no change was visible in the X-ray structures. There was a linear correlation between ?Tand total cavity volume, predicting that the cavity volume or atomic packing is an important factor regulating volume fluctuations during the molecular evolution of this protein.
Highlights
? Compressibility of evolutionary lysozymes are calculated by normal mode analysis. ? Volume fluctuation of lysozymes follows a nonneutral evolutionary pathway. ? Internal cavity is an important factor regulating volume fluctuation of lysozymes.
Publication year: 2011 Source: Biophysical Chemistry, Available online 3 December 2011 Kan Xiong, Lu Ma, Sanford A. Asher CD and UV resonance Raman measurements surprisingly find that the charge screening of even 2 M concentrations of NaCl and KCl do not alter the unfolded PPII and 2.51-helix conformations of poly-L-glutamate. These salts appear to be excluded from the region between the side chain charges and the peptide backbone. Furthermore, no direct ion pairing occurs between these salts and the side chain carboxylates.
Highlights
? Poly-L-glutamate adopts unfolded PPII and 2.51-helix conformations in pure water. ? Charge screening of 2 M salts do not alter the conformation of poly-L-glutamate. ? Salts are excluded from the region between side chain charges and peptide backbone. ? No direct ion pairing occurs between salts and the side chain carboxylates.
Publication year: 2011 Source: Biophysical Chemistry, Available online 1 December 2011 David L. Beauchamp, Mazdak Khajehpour Salt ions affect protein stability in a variety of ways. In general, these effects have either been interpreted from a charge solvation/charge screening standpoint or they have been considered to be the result of ion-specific interactions with a particular protein. Recent theoretical work suggests that a major contribution to salt effects on proteins is through the interaction of salt ions that are located near the protein surface and their induced point image charges that are located in the low-dielectric protein cavity. These interactions form the basis of “salting-out” interactions. Salt ions induce an image charge of the same sign in the low dielectric protein medium. The interaction between the induced charge and its mirror charge is repulsive and consequently thermodynamically destabilizing. However, a folded protein that has a much smaller surface area will be less destabilized than the unfolded state. Consequently, the folded state will be stabilized relative to the unfolded state. This work analyzes salt effects in the model enzyme Ribonuclease t1, and demonstrates that interactions between salt ions and their induced point charges provide a major contribution to the observed salt-induced increase in protein stability. This work also demonstrates that in the case of weakly-binding ions (ions with binding constants that are in the order of 50 Mand less), salting-out effects should still be considered in order to provide a more realistic interpretation of ion binding. These results should therefore be considered when salt effects are used to analyze electrostatic contributions to protein structure or are used to study the thermodynamics of proteins associated with halophillic organisms.
Highlights
? A major contribution to salt effects on proteins is through the interaction of salt ions that are located near the protein surface and their induced point image charges that are located in the low-dielectric protein cavity. ? Salt ions induce an image charge of the same sign in the low dielectric protein medium. The interaction between the induced charge and its mirror charge is repulsive and consequently thermodynamically destabilizing. ? Salt effects cause the folded protein state that has a much smaller surface area to be less destabilized than the unfolded state. Consequently, the folded state will be stabilized relative to the unfolded state. ? This work analyzes salt effects in the model enzyme Ribonuclease t1, and demonstrates that interactions between salt ions and their induced point charges provide a major contribution to the observed salt-induced increase in protein stability.
Publication year: 2011 Source: Biophysical Chemistry, Available online 25 November 2011 Mikhail Sergeev, Jody L. Swift, Antoine G. Godin, Paul W. Wiseman Fluorescence microscopy is widely used in the life sciences, but largely for qualitative imaging. Here we apply a bioanalytical technique, fluorescence image moment analysis, to demonstrate how the distribution of the fluorescent molecules can be measured directly from confocal microscopy images. We measured the oligomerization state of EGF-eGPF receptors expressed in CHO-K1 cellsin situ.
Highlights
? Fluorescence image moment analysis is capable of measuring the oligomerization distribution directly from confocal images ? Pharmacodynamic experiments of ligand-induced EGFR activation were carried out ? SpIDA revealed an increase of EGFR dimer density at early activation times. ? With ligand accessibility studies, we showed that the equilibrium between down-regulation and internalization is defined by the total ligand percent occupancy.
Publication year: 2011 Source: Biophysical Chemistry, Available online 17 November 2011 Manuel Montenegro, Laura Masgrau, Àngels González-Lafont, José M. Lluch, Mireia Garcia-Viloca cAMP-dependent protein kinase (PKA) is one of the simplest and best understood members of the protein kinase family. In a previous study, we have theoretically studied the complex between PKA and the heptapeptide substrate Kemptide by classical molecular dynamics. On the basis of the results obtained for Kemptide, the aim of the present work is to explore how the different conditions, such as phosphorylation state, substrate, and mutations of key residues affect the enzyme dynamics. We have built different models of the complex; particularly we have focused our attention on two crystallographic structures which main difference consists in their phosphorylation state. The first one has the residue Thr197 modified into a phospho-threonine (pThr197); the second one, in addition to the same Thr197, has also the residue Ser338 modified into a phospho-serine (pSer338). In addition, we have analyzed the effect of the choice of the substrate by building a model of the PKA-SP20 Michaelis complex. Finally, we have theoretically studied the effect of the mutation of the highly conserved residue Asp166 that, experimentally, leads to a decrease of the reaction rate. The results of this study give insight into the dynamical states of the enzyme and their relationship with different elements of the model, which correspond to different natural or human guided situations of the active biological system.
Highlights
? The Thr197 phosphorylation site seems to be necessary for the enzyme function. ? The different phosphorylation states influence the flexibility of the structure. ? Asp166 is crucial for the correct placement of the P-site of the substrate ? Asp 166 has an active role in limiting the accessibility to the active centre.
Publication year: 2011 Source: Biophysical Chemistry, Available online 10 November 2011 Laurence A. Belfiore, Michael L. Floren, Carol J. Belfiore This research contribution addresses electric-field stimulation of intra-tissue mass transfer and cell proliferation in viscoelastic biomaterials. The unsteady state reaction–diffusion equation is solved according to the von Kármán-Pohlhausen integral method of boundary layer analysis when nutrient consumption and tissue regeneration occur in response to harmonic electric potential differences across a parallel-plate capacitor in adielectric-sandwichconfiguration. The partial differential mass balance with diffusion and electro-kinetic consumption contains the Damköhler (?) and Deborah (De) numbers. Zero-field and electric-field-sensitive Damköhler numbers affect nutrient boundary layer growth. Diagonal elements of the 2nd-rank diffusion tensor are enhanced in the presence of weak electric fields, in agreement with the formalism of equilibrium and nonequilibrium thermodynamics. Induced dipole polarization density within viscoelastic biomaterials is calculated via the real and imaginary components of the complex dielectric constant, according to the Debye equation, to quantify electro-kinetic stimulation. Rates of nutrient consumption under zero-field conditions are described by third-order kinetics that include local mass densities of nutrients, oxygen, and attached cells. Thinner nutrient boundary layers are stabilized at shorter dimensionless diffusion times when the zero-field intra-tissue Damköhler number increases above its initial-condition-sensitive critical value [i.e., {?zero-field}critical ? 53, see Eq. (23)], such that the biomaterial core is starved of essential ingredients required for successful proliferation. When tissue regeneration occurs above the critical electric-field-sensitive intra-tissue Damköhler number, the electro-kinetic contribution to nutrient consumption cannot be neglected. The critical electric-field-sensitive intra-tissue Damköhler number is proportional to the Deborah number. Schematic representation of cylindrical biomaterials in a dielectric-sandwich configuration subjected to harmonic electric potential differences across the capacitor plates, with radial diffusion of nutrients inward to support cell proliferation and sustainability.
Highlights
? reaction–diffusion equation is analyzed in electric-field-stimulated porous biomaterials ? critical intra-tissue Damköhler number is quantified in the presence of an electric potential ? Damköhler and Deborah numbers quantify electric-field-stimulated chemical kinetics ? mass transfer boundary layer thickness depends on the Damköhler and Deborah numbers
Publication year: 2011 Source: Biophysical Chemistry, Available online 30 October 2011 Huan Zhan, Themis Lazaridis The implicit membrane model IMM1 is extended to include the membrane dipole potential and applied to molecular dynamics simulations of the helical peptides alamethicin, WALP23, influenza hemagglutinin fusion peptide, HIV fusion peptide, magainin, and the pre-sequence of cytochromecoxidase subunit IV (p25). The results show that the orientation of the peptides in the membrane can be influenced by the dipole potential. The binding affinity of all peptides except for the hemagglutinin fusion peptide decreases upon increase of the dipole potential. The changes in both orientation and binding affinity are explained by the interaction of the dipole potential with the helix backbone dipole and ionic side-chains. In general, peptides that tend to insert the N-terminus in the membrane and/or have positively charged side chains will lose binding affinity upon increase of the dipole potential.
Highlights
? IMM1 was extended to include the membrane dipole potential. ? The modified IMM1 was applied to MD simulations of helical peptides. ? Peptide orientation and binding affinity is affected by the dipole potential. ? This is due to interaction between the dipole potential and helix backbone dipole and/or ionic side-chains
Publication year: 2011 Source: Biophysical Chemistry, Available online 25 October 2011 Nisha Patel, David N. Dubins, Régis Pomès, Tigran V. Chalikian Partial molar volume, V°, has been used as a tool to sample solute hydration for decades. The efficacy of volumetric investigations of hydration depends on our ability to reliably discriminate between the cavity, VC, and interaction, VI, contributions to the partial molar volume. The cavity volume, VC, consists of the intrinsic volume, VM, of a solute molecule and the thermal volume, VT, with the latter representing the volume of the effective void created around the solute. In this work, we use molecular dynamics simulations in conjunction with the Kirkwood-Buff theory to compute the partial molar volumes for organic solutes of varying size in water. We perform our computations using the Lennard-Jones and Coulombic pair potentials as well as truncated potentials which contain only the Lennard-Jones but not the Coulombic contribution. The partial molar volume computed with the Lennard-Jones potentials in the absence of the Coulombic term nearly coincides with the cavity volume, VC. We determine the thermal volume, VT, for each compound by subtracting its van der Waals volume, VW, from VC. Finally, we apply the spherical approximation of solute geometry to evaluate the thickness of the thermal volume, ?. Our results reveal an increase in the thickness of thermal volume, ?, with an increase in the size of the solute. This finding may be related to dewetting of large nonpolar solutes and the concomitant increase in the compressibility of water of hydration.
Highlights
? We use MD simulations to compute the partial molar volumes for solutes of varying size. ? We determine the thermal volume, VT, for each compound. ? The thickness of thermal volume increases sigmoidally with the radius of a solute.
Publication year: 2011 Source: Biophysical Chemistry, Available online 29 September 2011 Jens Smiatek, Rakesh Kumar Harishchandra, Oliver Rubner, Hans-Joachim Galla, Andreas Heuer We have performed Molecular Dynamics simulations of ectoine, hydroxyectoine and urea in explicit solvent. Special attention has been spent on the local surrounding structure of water molecules. Our results indicate that ectoine and hydroxyectoine are able to accumulate more water molecules than urea by a pronounced ordering due to hydrogen bonds. We have validated that the charging of the molecules is of main importance resulting in a well defined hydration sphere. The influence of a varying salt concentration is also investigated. Finally we present experimental results of a DPPC monolayer phase transition that validate our numerical findings.
Highlights
? Investigation of solvent structure around compatible solutes via Molecular Dynamics simulations. ? Numerical study validates strong hygroscopic effect of ectoines. ? Long range influence on water structure is indicated. ? Independent on salt concentration. ? Qualitative agreement with presented experiments.
Publication year: 2011 Source: Biophysical Chemistry, Available online 24 September 2011 Yoshiteru Yonetani, Hidetoshi Kono The lifetime during which a water molecule resides at the surface of a biomolecule varies according to the hydration site. What determines this variety of lifetimes? Despite many previous studies, there is still no uniform picture quantitatively explaining this phenomenon. Here we calculate the lifetime for a particular hydration pattern in the DNA minor groove, the water bridge, for various DNA sequences to show that the water-bridge lifetime varies from 1 to ~ 300 ps in a sequence-dependent manner. We find that it follows 1/k(Vstep)Pm, wherePmandVstepare two crucial factors, namely the probability of forming a specific hydrogen bond in which more than one donor atom participates, and the structural fluctuation of DNA, respectively. This relationship provides a picture of the water kinetics with atomistic detail and shows that water dissociation occurs when a particular hydrogen-bonding pattern appears. The rate constant of water dissociationkcan be described as a function of the structural fluctuations of DNA. This picture is consistent with the model of Laage and Hynes proposing that hydrogen-bond switching occurs when an unusual number of hydrogen bonds are formed. The two new factors suggested here are discussed in the context of the surface's geometry and electrostatic nature, which were previously proposed as the determinants of water lifetimes.
Highlights
? Lifetime of water molecules in the DNA minor groove widely varies for various DNA sequences. ? Variety of the lifetime is associated with the formation of a specific hydrogen bond and structural fluctuations of DNA basepair step. ? The water kinetics is consistent with the hydrogen-bond switching model of Laage and Hynes. ? Lifetime of water molecules in the DNA minor groove widely varies for various DNA sequences. ? Variety of the lifetime is associated with the formation of a specific hydrogen bond and structural fluctuations of DNA basepair step. ? The water kinetics is consistent with the hydrogen-bond switching model of Laage and Hynes.
Publication year: 2011 Source: Biophysical Chemistry, Available online 21 September 2011 Petra Pullmannová, Margarida Bastos, Guangyue Bai, Sergio S. Funari, Ivan Lacko, ... Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-?,?-diyl-bis(alkyldimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L?). In addition to theL?phase, a coexisting lamellar phaseLBwas also identified in the complexes formed, with periodicities in the range ~ 8.8 – 5.7 nm, at ionic strengths corresponding to 50 – 200 mM NaCl. The periodicities ofLBphase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.
Publication year: 2011 Source: Biophysical Chemistry, Available online 19 September 2011 Paul Y. Kim, Michael E. Nesheim In an attempt to resolve the ongoing debate regarding the mechanism of PT activation by prothrombinase, Lee and coworkers (Biophys Chem 2010, 149:28–33) consolidated the empirical data reported by the Nesheim (JBC 2007, 282:32568–91) and Krishnaswamy (JBC 2004, 279:54927–36.) groups, and used this information to generate average time course data for PT and its intermediates. A modified mathematical model, originally reported by the Nesheim group, then was fit to the 'average' data. Based on their analysis, the authors conclude that a one-form model adequately describes the time course data, thereby rendering channeling or ratcheting unnecessary. We believe that the analysis proposed by these authors is flawed because a) it averages data based on experiments done under different conditions, and b) the levels of meizothrombin were included twice when determining the total concentration of prothrombin species. When we recalculated our data using the equation suggested by Lee and colleagues, we failed to confirm their findings. Based on our analysis, channeling alone or both channeling and racheting are necessary to fit the time course data. Therefore, we disagree with the conclusions of Lee and colleagues and we believe that our previous findings still hold.
Publication year: 2011 Source: Biophysical Chemistry, Available online 19 September 2011 Chang Jun Lee, Sangwook Wu, Lee G. Pedersen We analyze the data of Kim and Nesheim for the catalytic conversion of prothrombin to thrombin. We do not reach the same conclusions of Kim and Nesheim in the preceding letter. We find that based on the Kim-Nesheim data, the one-form model (which involves 12 or 22 rate constants) cannot be rejected. Further, we find that a simpler four constants model captures the functional dependence of the concentration profiles of the four participants: prothrombin (PT), meizothrombin (M), prethrombin-2 (Pre-2), and thrombin (T). The simple model can also be interpreted in terms of a recent solvent-equilibrated, all-atom structural model of the reaction complex.
Publication year: 2011 Source: Biophysical Chemistry, Available online 17 September 2011 Marco Frasconi, Silvia Chichiarelli, Elisa Gaucci, Franco Mazzei, Caterina Grillo, ... The protein ERp57 (also known as PDIA3) is a widely distributed protein, mainly localized in the endoplasmic reticulum, where it acts as disulfide isomerase, oxidoreductase and chaperone, in concert with the lectins calreticulin (CRT) and calnexin. The ERp57/CRT complex has been detected on the cell surface and previous studies have suggested its involvement in programmed cell death. Although the ERp57-CRT complex has been characterized, little is known about its role in different cellular compartments as well as inhibitors of this interaction.We focused on the kinetic, extent and stability of the ERp57-CRT complex, using the surface plasmon resonance spectroscopy, investigating the possible role as inhibitor of the antibiotic vancomycin. Equilibrium thermodynamic data suggested that vancomycin may hinder the interaction between the two proteins and could interfere with the ERp57 conformational changes that stabilize the complex. Furthermore, by means of confocal microscopy, we evaluated the effect of thein vivoadministration of vancomycin on the ERp57/CRT complex on the surface of HeLa cells.The model presented here could be used for the search of other specific inhibitors/interactors of ERp57, which can be extremely helpful to understand the biological pathways where the protein is involved and to modulate its activity. Sensorgrams for the affinity interaction of variable concentration of CRT to immobilized ERp57 in the presence of vancomycin. The concentrations of calreticulin (?g ml) were: 0.5 (a), 1.0 (b), 2.0 (c), 3.0 (d) and 4.0 (e).
Highlights
? SPR data provide evidence for an ERp57 conformational change in the binding to CRT. ? Vancomycin could hinder the stabilizing conformational change in ERp57-CRT complex. ? Vancomycin decreases the amount of calreticulin on plasma membrane.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 119-125 Xiujun Jiao, Eleonora Keating, Seyed Tadayyon, Fred Possmayer, Yi Y. Zuo, ... Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air–liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function.
Highlights
?This study analyzes the film structure of endogenous rat surfactant. ?Phase separation within the surfactant film occurs at low surface pressures. ?Film structure changes when surface pressure increases, leading to multilayers. ?Multilayer formation appears to represent the critical functional process.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 150-156 Dejan Gmajner, Pegi Ahlin Grabnar, Magda Tušek Žnidari?, Jasna Štrus, Marjeta Šentjurc, ... The physicochemical properties of binary lipid mixtures of diether C25,25lipids and dipalmitoyl-L-?-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol% C25,25lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.
Highlights
? Binary mixture consists of archaeal diether and DPPC lipids can form liposomes. ? Higher content of archaeal lipids in liposomes decrease the permeability and the fluidity. ? Mixed liposomes do not fuse or aggregate after storage for 41 day at low temperature. ? The mixed liposomes have the potential to be used as a new drug-delivery system.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 54-60 Ruiting Zhang, Tiejun Xiao, Zhonghuai Hou The effect of internal noise in a delayed circadian oscillator is studied by using both chemical Langevin equations and stochastic normal form theory. It is found that internal noise can induce circadian oscillation even if the delay time?is below the deterministic Hopf bifurcation?h. We use signal-to-noise ratio (SNR) to quantitatively characterize the performance of such noise induced oscillations and a threshold value of SNR is introduced to define the so-called effective oscillation. Interestingly, the?-range for effective stochastic oscillation, denoted as??EO, shows a bell-shaped dependence on the intensity of internal noise which is inversely proportional to the system size. We have also investigated how the rates of synthesis and degradation of the clock protein influence the SNR and thus??EO. The decay rateKdcould significantly affect??EO, while varying the gene expression rateKehas no obvious effect ifKeis not too small. Stochastic normal form analysis and numerical simulations are in good consistency with each other. This work provides us comprehensive understandings of how internal noise and time delay work cooperatively to influence the dynamics of circadian oscillations.
Research Highlights
? We study constructive role of noise in a delayed circadian oscillator. ? Internal noise can enhance the range for effective oscillation. ? Effective oscillation range shows maximum at optimal system size. ? Theoretical analysis successfully uncovers the mechanism. ? The phenomena are quite robust to parameter changes.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 134-140 Ilaria Rivolta, Alice Panariti, Maddalena Collini, Barbara Lettiero, Laura D'Alfonso, ... We have measured in human alveolar cells the cytoplasmic distribution of the fluorophore coumarin-6 carried by Solid Lipid Nanoparticles (SLNs) and observed a perinuclear accumulation of the fluorescence that can be described by a single exponential growth along an ideal line joining the plasma membrane to the nuclear border and by a sigmoidal relationship as a function of time. Intracellular distribution was affected by lowering the temperature from 37 to 4 °C and by the disruption of cytoskeleton by cytochalasin D, but it was minimally perturbed by the inhibition of ATP dependent molecular motors. A biophysical model was developed for an accumulation of loaded particles against a diffusion gradient based on a mean field interaction energy, whose origin we ascribe to the actin structure of the cytoskeleton. The estimated value for the load diffusion coefficient was four and two orders of magnitude less than that of free coumarin-6 and of SLNs in aqueous solutions, respectively, suggesting that the load moves within the cell cytoplasm in a form still reminiscent of the nanocarrier structure.
Highlights
? We have measured the cytoplasmic distribution of the coumarin-6 carried by Solid Lipid Nanoparticles in human alveolar cells. ? Observed a perinuclear accumulation of the fluorescence that we model with the a mean field interaction of the load with the cytoskeleton. ? Experiments as a function of temperature, the cytoskeleton state and metabolic poisons validate this analysis.
Publication year: 2011 Source: Biophysical Chemistry, Volume 159, Issue 1, November 2011, Pages 217-226 Peregrine Bell-Upp, Aaron C. Robinson, Steven T. Whitten, Erika L. Wheeler, Janine Lin, ... The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential Hbinding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pKavalues in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pKavalues in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pKavalues of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.
Highlights
? Acid unfolding of proteins is governed by Asp and Glu residues that have different pKavalues in the native and unfolded states. ? Proteins are unfolded by acid because Asp and Glu residues tend to have depressed pKavalues in the native state and normal ones in the unfolded state. ? Substantial manipulation of the acid unfolding of a protein by direct manipulation of Asp and Glu residues is extremely challenging. ? The most efficient way to alter the acid unfolding profile of a protein is through manipulation of global stability with mutagenesis, osmolytes or salt. ? The molecular mechanism of the acid unfolding process for a protein can be different at different pH values.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 111-118 M. Bolean, A.M.S. Simão, B.Z. Favarin, J.L. Millán, P. Ciancaglini Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with “lipid rafts”, ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc = 41.5 °C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ?H and ?t1/2values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP. DSC thermograms of quaternary systems (10 mg/mL). Differential scanning calorimetry thermograms were processed in excess heat capacity, Cp (kcal·K·mol) as a function of temperature (°C) of vesicles constituted by DPPC:Chol:SM:GM1 (7:1:1:1), molar ratio: (A) Liposome and (B) Proteoliposome. Dashed curves symbolize deconvolution analysis.
Research highlights
? The formed liquid-ordered complexes in the liposomes present a great lipid packing. ? A pronounced decrease in ?H when liposome system is compared with proteoliposome. ? Enzyme incorporation influences the reduction in cooperativity, but no changes in TC. ? TNAP presence resulted in an even greater segregation of the phase transition peaks. ? These systems are excellent to study changes in enzyme activity in model membranes.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 21-25 Peter R. Wills, Donald J. Winzor Theory is developed for the characterization of protein interactions by spectral techniques, where the constraints of constant temperature and pressure demand that thermodynamic activity be defined on the molal concentration scale. The customary practice of defining the equilibrium constant (K) on a molar basis is accommodated by developing expressions to convert those experimental values (Kmolar) to their thermodynamically more rigorous counterparts (Kmolal). Such procedures are illustrated by reanalysis of published results for the effects of molecular crowding agents on the isomerisation of ?-chymotrypsin and reversible complex formation between catalase and superoxide dismutase. Although those reanalyses have led to only minor refinements of the quantitative interpretation, it is clearly preferable to adopt thermodynamic rigor throughout future spectral studies by employing the molal concentration scale from the outset.
Research Highlights
? Molal concentrations should be used to characterize reactions by spectral methods. ? Rigorous expressions are adapted to accommodate experimental use of the molar scale. ? Results for the effect of sucrose on ?-chymotrypsin isomerization are reinterpreted. ? Crowding effects on catalase?superoxide dismutase interactions are reexamined. ? Future spectral studies should adopt the molal concentration scale from the outset.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 126-133 Defang Ouyang, Hong Zhang, Harendra S. Parekh, Sean C. Smith Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene deliveryin vivo. Previous reports based onin vitrostudies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing ‘late-endosomes’. Our simulations reveal that the time taken for the dendrimer–gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer–nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer–siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the “proton sponge theory”, thereby advancing the rational design of non-viral gene delivery systems. This figure is the snapshot of 14dendrimer complexed in the minor groove of RNA in 10 ns of MD simulation.
Research highlights
? We adopt molecular dynamics simulations to compare the complexation of siRNA with dendrimers at different pHs. ? Our simulations reveal that more compact packaging of the dendriplex at low pH than that at neutral pH. ? These novel simulations provide a detailed understanding of polymer-siRNA behaviour in the endosomal environment.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 141-149 Anna Niedzwiecka, Malgorzata Lekka, Per Nilsson, Anders Virtanen Deadenylation is the initial and often rate-limiting step in the main pathways of eukaryotic mRNA decay. Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that efficiently degrades mRNA poly(A) tails. Structural and functional studies have shown that human PARN is composed of at least three functional domains,i.e.the catalytic nuclease domain and two RNA binding domains, the R3H and the RNA recognition motif (RRM), respectively. However, the complete structure of the full length protein is still unknown. We have investigated the global architecture of human PARN by atomic force microscopy (AFM) imaging in buffered milieu and report for the first time the dimensions of the full length protein at subnanometer resolution. The AFM images of single PARN molecules reveal compact ellipsoidal dimers (10.9 × 7.6 × 4.6 nm). The dimeric form of PARN was confirmed by dynamic light scattering (DLS) measurements that rendered a molecular weight of 161 kDa, in accordance with previous crystal structures of PARN fragments showing a dimeric composition. We discuss a putative internal arrangement of three functional domains within the full length PARN dimer.
Research highlights
? AFM images show that a single PARN molecule form a compact ellipsoidal dimer. ? Dimensions of the single protein molecule were determined as 10.9 × 7.6 × 4.6 nm. ? DLS was used to further characterize PARN and its fragment encompassing the RRM domain.
Publication year: 2011 Source: Biophysical Chemistry, Available online 10 September 2011 Zheng Yi, Michihiro Nagao, Dobrin P. Bossev The effect of the charged lidocaine on the structure and dynamics of DMPC/DMPG (mass fraction of 95/5) unilamellar vesicles has been investigated. Changes in membrane organization caused by the presence of lidocaine were detected through small angle neutron scattering experiments. Our results suggest that the presence of lidocaine in the vicinity of the headgroups of lipid membranes leads to an increase of the area per lipid molecule and to a decrease of membrane thickness. Such changes in membrane structure may induce disordering of the tail group. This scenario explains the reduction of the main transition temperature of lipid membranes, as the fraction of lidocaine per lipid molecules increases, which was evident from differential scanning calorimetry results. Furthermore neutron spin echo spectroscopy was used for the dynamics measurements and the results reveal that presence of charged lidocaine increases the bending elasticity of the lipid membranes in the fluid phase and slows the temperature-dependent change of bending elasticity across the main transition temperature.
Highlights
? We investigated the effect of lidocaine on the structure and dynamics of DMPC/DMPG membranes ? Presence of lidocaine leads to the increase of the area per lipid molecule and the consecutive decrease of the membrane thickness ? Change in the membrane structure induces depression of the tail group ordering of the lipid molecules ? Lidocaine increases the bending elasticity of DMPC/DMPG lipid membranes in liquid crystalline phase and reduces the temperature-dependence of bending elasticity across the main transition temperature
Publication year: 2011 Source: Biophysical Chemistry, Volume 159, Issue 1, November 2011, Pages 210-216 Jianquan Li, J. Ching Lee The communication mechanism(s) responsible for the allosteric behavior of E.coli cAMP binding receptor protein, CRP, is still a subject of intense investigation. As a tool to explore the communication mechanism, the mutations at various positions in the cAMP-binding (K52N, D53H, S62F and T127L) or the DNA- binding (H159L) domain or both (K52N/H159L) were generated. The sites and specific nature of side chain substitutions were defined by earlier genetic studies, the results of which show that these mutants have a similar phenotype i.e. they are activated without exogenous cAMP. Presently, no significant changes in the structures of WT and mutant CRPs have been observed. Hence, the pressing issue is to identify a physical parameter that reflects the effects of mutations. In this study, the stability of these various CRP species in the presence of GuHCl was monitored by three spectroscopic techniques, namely, CD, tryptophan fluorescence and FT-IR which could provide data on the stability of ?-helices and ?-strands separately. Results of this study led to the following conclusions: 1. The ?-helices can be grouped into two families with different stabilities. Mutations exert a differential effect on the stability of helices as demonstrated by a biphasic unfolding curve for the helices. 2. Regardless of the locations of mutations, the effects can be communicated to the other domain resulting in a perturbation of the stability of both domains, although the effects are more significantly expressed in the stability of the helices. 3. Although in an earlier study [Gekko, et al. Biochemistry 43 (2004) 3844] we showed that cooperativity of cAMP binding is generally correlated to the global dynamics of the protein and DNA binding affinity, in this study we found that generally there is no clear correlation between functional energetics and stability of secondary structures. Thus, results of this study imply that modulation of allostery in CRP is entropic in nature. Effect of mutation on structural stability
Highlights
? Mutations exert a differential effect on the stability of helices. ? The effects of mutations can be communicated to the sheets. ? Cooperativity of cAMP binding is generally correlated to the stability of the sheets.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 91-95 I. Topol, J. Collins, A. Savitsky, A. Nemukhin Computational methods of quantum chemistry are used to characterize structures and vertical excitation energies of the S0–S1optical transitions in the chromophore binding pockets of the red fluorescent proteins DsRed and of its artificial mutant mCherry. As previously shown, optimizing the equilibrium geometry configurations with B3LYP density functional theory, followed by ZINDO calculations of the electronic excitations, yields positions of the optical bands in good agreement with experimental data. These large scale quantum calculations elucidate the role of the hydrogen bonded network as well as point mutations in the absorption spectra of the DsRed and mCherry proteins. The effect of an external electric field applied to the fluorescent protein chromophores is examined and shows that such fields may result in large shifts in spectral bands. These strategies can be applied for rational design of the fluorescent proteins by site-directed mutagenesis.
Research Highlights
? Quantum chemical methods were used to characterize structures and absorption spectra of the red fluorescent proteins. ? The role of the hydrogen bond network and point mutations in the chromophore binding pockets was investigated. ? The effect of an external electric field applied to the fluorescent protein chromophores was examined.
Publication year: 2011 Source: Biophysical Chemistry, Available online 9 September 2011 Eleanor Y.M. Bonnist, Kirsten Liebert, David T.F. Dryden, Albert Jeltsch, Anita C. Jones The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.
Highlights
? The anomalous response of 2AP intensity to M.EcoRV binding is resolved by measuring the fluorescence decay. ? The fluorescence decay parameters of 2AP report unambiguously and informatively on DNA conformation and the DNA-enzyme interface. ? When M.EcoRV binds to its recognition sequence it induces different local distortions at the two adenine positions. ? The target 2AP is flipped, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site. ? Bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 1-8 Laurence A. Belfiore, Michael L. Floren, Alexandre T. Paulino, Carol J. Belfiore This research contribution addresses the mechanochemistry of intra-tissue mass transfer for nutrients, oxygen, growth factors, and other essential ingredients that anchorage-dependent cells require for successful proliferation on biocompatible surfaces. The unsteady state reaction–diffusion equation (i.e., modified diffusion equation) is solved according to the von Kármán–Pohlhausen integral method of boundary layer analysis when nutrient consumption and tissue regeneration are stimulated by harmonically imposed stress. The mass balance with diffusion and stress-sensitive kinetics represents a rare example where the Damköhler and Deborah numbers appear together in an effort to simulate the development of mass transfer boundary layers in porous viscoelastic biomaterials. The Boltzmann superposition integral is employed to calculate time-dependent strain in terms of the real and imaginary components of dynamic compliance for viscoelastic solids that transmit harmonic excitation to anchorage-dependent cells. Rates of nutrient consumption under stress-free conditions are described by third-order kinetics which include local mass densities of nutrients, oxygen, and attached cells that maintain dynamic equilibrium with active protein sites in the porous matrix. Thinner nutrient mass transfer boundary layers are stabilized at shorter dimensionless diffusion times when the stress-free intra-tissue Damköhler number increases above its initial-condition-sensitive critical value. The critical stress-sensitive intra-tissue Damköhler number, above which it is necessary to consider the effect of harmonic strain on nutrient consumption and tissue regeneration, is proportional to the Deborah number and corresponds to a larger fraction of the stress-free intra-tissue Damköhler number in rigid biomaterials. von Kármán–Pohlhausen boundary layer predictions for nutrient diffusion and stress-sensitive consumption in porous biomaterials that experience viscoelastic relaxation (i.e., De = 1)
Research Highlights
?Reaction-diffusion equation:is analyzed in porous biomaterials that contain anchorage-dependent cells subjected to harmonic tensile stress. ?Critical intra-tissue Damköhler number:ratio of nutrient consumption rate relative to the rate of intra-tissue diffusion is quantified in the presence of mechanical stress. ?Boltzmann superposition integral:is employed to calculate time-dependent strain in rigid and mobile viscoelastic biomaterials. ?Effect of viscoelastic relaxation on chemical kinetics:time-dependent strain is used to modify reaction rates in stress-sensitive systems. ? von Kármán nutrient boundary layer thickness is calculated to estimate the time required for regenerative tissue growth.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 96-103 Mingjuan Wang, Renee D. JiJi The mechanism by which flavonoids prevent formation of amyloid-? (A?) fibrils, as well as how they associate with non-fibrillar A? is still unclear. Fresh, un-oxidized myricetin exhibited excitation and emission fluorescence maxima at 481 and 531 nm, respectively. Introduction of either A?(1–42) or A?(25–40) resulted in a fluorescence decrease, when measured at 481 nm, suggesting formation of a myricetin–A? complex. Circular dichroism (CD) and ultraviolet resonance Raman (UVRR) studies indicate that the association of myricetin with the A? peptide or its hydrophobic fragment, A?(25–40), leads to subtle changes in each peptide's conformation. A?(25–40) formed amyloid fibrils at a similar rate, when compared to the full-length peptide, A?(1–42), using thioflavin T (ThT) fluorescence. Studies also indicated that myricetin was equally effective at preventing the formation of both A?(1–42) and A?(25–40) fibrils. Although ThT assays indicated that A?(1–16) did not form amyloid fibrils, CD studies of the hydrophilic fragment, A?(1–16), suggest possible interactions between myricetin and aromatic side chains. UVRR studies of the full-length peptide and A?(1–16) showed increases in the intensity of the aromatic modes upon introduction of myricetin. Our findings suggest that myricetin interacts with soluble A? via two mechanisms, association with the hydrophobic C-terminal region and interactions with the aromatic side chains.
Highlights
? The short hydrophobic peptide, A?(25–40), exhibits a similar aggregation propensity to the full-length A? peptide. ? The flavonoid myricetin inhibits A?(1–42) and A?(25–40) fibril formation. ? Deep-UVRR studies indicate that myricetin may interact with aromatic residues of A?. ? CD, UVRR and fluorescence studies indicate non-aromatic myricetin–A? interactions at the hydrophobic C-terminus.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 9-20 Maria J. Jezewska, Michal R. Szymanski, Wlodzimierz Bujalowski Kinetic mechanism of the ssDNA recognition by the polymerase X of African Swine Fever Virus (ASFV) and energetics of intermediate formations have been examined, using the fluorescence stopped-flow method. The association is a minimum three-step processThe nucleic acid makes the initial contact through the C-terminal domain, which generates most of the overall ?G°. In the second step the nucleic acid engages the N-terminal domain, assuming the bent structure. In equilibrium, the complex exists in at least two different states. Apparent enthalpy and entropy changes, characterizing formations of intermediates, reflect association of the DNA with the C-terminal domain and gradual engagement of the catalytic domain by the nucleic acid. The intrinsic DNA-binding steps are entropy-driven processes accompanied by the net release of water molecules. The final conformational transition of the complex does not involve any large changes of the DNA topology, or the net release of the water molecules.
Research Highlights
?The ASFV pol X–ssDNA association is three-step sequential mechanism. ?The enzyme makes first contact with the DNA through its C-terminal domain. ?Engagement of the catalytic domain induces large topological changes of the DNA. ?Intrinsic binding steps are entropy-driven and accompanied by the release of similar numbers of water molecules. ?The final transition does not affect the bound DNA structure.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 38-45 Yoon Jung Jang, Ga-Young Yeo, Borami Park, Seog K. Kim The binding modes of the [Ru(II)(1,10-phenanthroline)(L1L2) dipyrido[3,2-a:2?,3?-c]phenazine]{[Ru(phen)(py) Cl dppz](L1 = Cl, L2 = pyridine) and ([Ru(phen)(py)2dppz](L1 = L2 = pyridine)} to native DNA is compared to that of the [Ru(II)(1,10-phenanthroline)2dipyrido[3,2-a:2?,3?-c]phenazine]complex ([Ru(phen)2dppz]) by various spectroscopic and hydrodynamic methods including electric absorption, linear dichroism (LD), fluorescence spectroscopy, and viscometric titration. All measured properties, including red-shift and hypochromism in the dppz absorption band, nearly perpendicular molecular plane of the dppz ligand with respect to the local DNA helix axis, prohibition of the ethidium binding, the light switch effect and binding stoichiometry, increase in the viscosity upon binding to DNA, increase in the melting temperature are in agreement with classical intercalation of dppz ligand of the [Ru(phen)2dppz]complex, in which both phenanthroline ligand anchored to the DNA phosphate groups by electrostatic interaction. [Ru(phen)(py)2dppz]and [Ru(phen)(py) Cl dppz]complexes had one of the phenanthroline ligand replaced by either two pyridine ligands or one pyridine plus a chlorine ion. They exhibited similar protection from water molecules, interaction with DNA bases, and occupying site that is common with ethidium. The dppz ligand of these two Ru(II) complex were greatly tilted relative to the DNA helix axis, suggesting that the dppz ligand resides inside the DNA and is not perpendicular relative to the DNA helix axis. These observation suggest that anchoring the [Ru(phen)2dppz]complex by both phenanthroline is essential for the dppz ligand to be classically intercalated between DNA base-pairs.
Research Highlights
? The dppz ligand of the [Ru(II)(phen)2dppz]complex intercalates between DNA base pairs. ? Replacing the phenanthroline by Cl and pyridine alters the spectral properties. ? Replacing the anchoring moieties resulted in alteration in the binding mode. ? Dppz ligand remains inside of DNA with a large tilt angle.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 26-37 Maria J. Jezewska, Michal R. Szymanski, Wlodzimierz Bujalowski Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. The primary DNA-binding subsite of the enzyme, independent of the DNA conformation, is located on the C-terminal domain. Association of the bound DNA with the catalytic N-terminal domain finalizes the engagement of the total DNA-binding site of the enzyme and induces a large topological change in the structure of the bound ssDNA. The free energy of binding includes a conformational transition of the protein. Large positive enthalpy changes accompanying the ASFV pol X–ssDNA association indicate that conformational changes of the complex are induced by the engagement of the N-terminal domain. The enthalpy changes are offset by large entropy changes accompanying the DNA binding to the C-terminal domain and the total DNA-binding site, predominantly resulting from the release of water molecules.
Research highlights
? The primary DNA-binding site of ASFV pol X is located on the C-terminal domain. ? The total free energy of binding includes communication between the C-terminal and catalytic domains. ? The pol X - ssDNA association is an entropy-driven process. ? Engagement of the N-terminal domain is accompanied by a large positive enthalpy change. ? Entropy change results from the water release accompanying the binding.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issues 2-3, October 2011, Pages 104-110 Javier Murciano-Calles, Eva S. Cobos, Pedro L. Mateo, Ana Camara-Artigas, Jose C. Martinez Equilibrium unfolding at neutral pH of the third PDZ domain of PSD95 is well described by the presence of a partly unfolded intermediate that presents association phenomena. After some days' incubation annular and fibrillar structures form from the oligomers. At pH values below 3, however, differential scanning calorimetry shows that PDZ3 seems to unfold under a two-state scheme. Kinetic measurements followed by dynamic light scattering, ThT and ANS fluorescence reveal that the misfolding pathway still exists despite the absence of any populated intermediates and shows an irreversible assembling of the supramacromolecular structures as well as an appreciable lag-phase, contrary to what is found in similar experiments at neutral pH. Moreover, as shown by transmission-electron-microscopy images, the annular structures seen at neutral pH completely disappear from incubated solutions. According to the structural information, this titration behavior appears to be the consequence of a conformational equilibrium that depends on the protonation of some Glu residues located at the C-terminal ?3 helix and at the hairpin formed by strands ?2 and ?3. Our calculations suggest that the enthalpic contribution of these interactions may well be as much as 40 kJ·mol. The possible regulatory role of this equilibrium upon PDZ3 functionality and amyloid formation is briefly discussed.
Research highlights
? Unfolding at pH > 3.5 of PSD95-PDZ3 reveals the presence of an equilibrium oligomeric intermediate state that self-associates into annular and fibrillar structures reversibly. ? At pH < 3 PDZ3 seems to unfold under a two-state scheme, although the misfolding pathway still exists. ? The supramacromolecular structures organize irreversibly and show a lag-phase at acidic pH. ? This titration behavior is due to a conformational equilibrium that depends on the protonation of some Glu residues. ? The enthalpic contribution of these interactions may well be as much as 40 kJ·mol.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 46-53 Paola Sassi, Alessandra Giugliarelli, Marco Paolantoni, Assunta Morresi, Giuseppe Onori The unfolding of hen egg-white lysozyme dissolved both in D2O and CH3CH2OD/D2O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365 K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event.
Research Highlights
? We followed the effects of ethanol addition to lysozyme/water solutions. ? A reduced stability of the folded state was revealed on increasing ethanol fraction. ? An enhancement of intermolecular interactions between protein chains was observed upon increase of alcohol concentration. ? We evidenced an irreversible association initiated by the unfolded and the folded states of the macromolecule. ? We obtained different hydrogels under different thermal treatment of a single protein solution.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 73-80 Nicholus Bhattacharjee, Parbati Biswas Hydration layer water molecules play important structural and functional roles in proteins. Despite being a critical component in biomolecular systems, characterizing the properties of hydration water poses a challenge for both experiments and simulations. In this context we investigate the local structure of hydration water molecules as a function of the distance from the protein and water molecules respectively in 188 high resolution protein structures and compare it with those obtained from molecular dynamics simulations. Tetrahedral order parameter of water in proteins calculated from previous and present simulation studies show that the potential of bulk water overestimates the average tetrahedral order parameter compared to those calculated from crystal structures. Hydration waters are found to be more ordered at a distance between the first and second solvation shell from the protein surface. The values of the order parameter decrease sharply when the water molecules are located very near or far away from the protein surface. At small water–water distance, the values of order parameter of water are very low. The average order parameter records a maximum value at a distance equivalent to the first solvation layer with respect to the water–water radial distribution and asymptotically approaches a constant value at large distances. Results from present analysis will help to get a better insight into structure of hydration water around proteins. The analysis will also help to improve the accuracy of water models on the protein surface.
Research highlights
? We have studied distribution of the tetrahedral order parameter of hydration waters in high resolution protein structures. ? Robustness of the results is validated by studying similar distribution in cryogenic proteins. ? Differences are pointed out by comparing the database results with molecular dynamics simulation results.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 61-72 S. Dutta Banik, N. Nandi The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase fromEscherichia coli,Thermus thermophilusandStaphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mgions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction forE. coli,T. thermophilusandS. aureusare calculated using the combinedab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.
Research highlights
? A QM/MM study of the aminoacylation reaction in three species is carried out. ? The reaction is observed to follow a common reaction pathway for the three species. ? Conserved structural patterns are noted in aminoacylation reaction. ? The mechanism is dependent on conserved active site residues in close proximity of the reactants.
Publication year: 2011 Source: Biophysical Chemistry, Available online 7 September 2011 Lyndsey R. Powell, Kyle D. Dukes, Robin K. Lammi One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-? peptide (A?). Given the deleterious effects reported for A? dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled A? peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2–5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of A? species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments.
Highlights
? Four fibrillogenesis inhibitors reduce zinc- and acid-promoted oligomerization. ? ?-structured inhibitors with blocking groups best prevent association at pH 5.8. ? With zinc, multiple strategies give equal inhibition; oligomers may be looser. ? N-methylated A?16-22 m is most effective across both sample environments.
Publication year: 2011 Source: Biophysical Chemistry, Volume 158, Issue 1, September 2011, Pages 81-89 Douglas J. Martin, Marina Ramirez-Alvarado Amyloid formation occurs when a precursor protein misfolds and aggregates, forming a fibril nucleus that serves as a template for fibril growth. Glycosaminoglycans are highly charged polymers known to associate with tissue amyloid deposits that have been shown to accelerate amyloidogenesisin vitro. We studied two immunoglobulin light chain variable domains from light chain amyloidosis patients with 90% sequence identity, analyzing their fibril formation kinetics and binding properties with different glycosaminoglycan molecules. We find that the less amyloidogenic of the proteins shows a weak dependence on glycosaminoglycan size and charge, while the more amyloidogenic protein responds only minimally to changes in the glycosaminoglycan. These glycosaminoglycan effects on fibril formation do not depend on a stable interaction between the two species but still show characteristic traits of an interaction-dependent mechanism. We propose that transient, predominantly electrostatic interactions between glycosaminoglycans and the precursor proteins mediate the acceleration of fibril formationin vitro.
Research highlights
? Two amyloidogenic Light chains show differing response to the presence of glycosaminoglycans. ? AL-103 shows a weak dependence on heparin size- and charge on fibril formation reactions. ? Acceleration effects observed do not depend on a stable interaction between GAGs and protein. ? GAG mediated acceleration of fibril formation shows concentration and salt dependence. ? We propose that transient interactions between GAGs and a precursor state drive fibril formation.
Posted on 11 September 2011 | 12:02 pm
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