Dendritic atrophy constricts functional maps in resonance and impedance properties of hippocampal model neurons

A gradient in the density of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels is necessary for the emergence of several functional maps within hippocampal pyramidal neurons. Here, we systematically analyzed the impact of dendritic atrophy on nine such functional maps, related to input resistance and local/transfer impedance properties, using conductance-based models of hippocampal pyramidal neurons. We introduced progressive dendritic atrophy in a CA1 pyramidal neuron reconstruction through a pruning algorithm, measured all functional maps in each pruned reconstruction, and arrived at functional forms for the dependence of underlying measurements on dendritic length. We found that, across frequencies, atrophied neurons responded with higher efficiency to incoming inputs, and the transfer of signals across the dendritic tree was more effective in an atrophied reconstruction. Importantly, despite the presence of identical HCN-channel density gradients, spatial gradients in input resistance, local/transfer resonance frequencies and impedance profiles were significantly constricted in reconstructions with dendrite atrophy, where these physiological measurements across dendritic locations converged to similar values. These results revealed that, in atrophied dendritic structures, the presence of an ion channel density gradient alone was insufficient to sustain homologous functional maps along the same neuronal topograph. We assessed the biophysical basis for these conclusions and found that this atrophy-induced constriction of functional maps was mediated by an enhanced spatial spread of the influence of an HCN-channel cluster in atrophied trees. These results demonstrated that the influence fields of ion channel conductances need to be localized for channel gradients to express themselves as homologous functional maps, suggesting that ion channel gradients are necessary but not sufficient for the emergence of functional maps within single neurons.

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca2+ ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 mu M and 120 mu M indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 mu M and 1.7 mu M. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a alpha-helical rich protein. Calcium binding further increased the alpha-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. (C) 2015 Elsevier GmbH. All rights reserved.

Comparative evaluation of inversion approaches of the radiative transfer model for estimation of crop biophysical parameters

The inversion of canopy reflectance models is widely used for the retrieval of vegetation properties from remote sensing. This study evaluates the retrieval of soybean biophysical variables of leaf area index, leaf chlorophyll content, canopy chlorophyll content, and equivalent leaf water thickness from proximal reflectance data integrated broadbands corresponding to moderate resolution imaging spectroradiometer, thematic mapper, and linear imaging self scanning sensors through inversion of the canopy radiative transfer model, PROSAIL. Three different inversion approaches namely the look-up table, genetic algorithm, and artificial neural network were used and performances were evaluated. Application of the genetic algorithm for crop parameter retrieval is a new attempt among the variety of optimization problems in remote sensing which have been successfully demonstrated in the present study. Its performance was as good as that of the look-up table approach and the artificial neural network was a poor performer. The general order of estimation accuracy for para-meters irrespective of inversion approaches was leaf area index > canopy chlorophyll content > leaf chlorophyll content > equivalent leaf water thickness. Performance of inversion was comparable for broadband reflectances of all three sensors in the optical region with insignificant differences in estimation accuracy among them.

Modelling the influence of land-use changes on biophysical and biochemical interactions at regional and global scales

Land-use changes since the start of the industrial era account for nearly one-third of the cumulative anthropogenic CO2 emissions. In addition to the greenhouse effect of CO2 emissions, changes in land use also affect climate via changes in surface physical properties such as albedo, evapotranspiration and roughness length. Recent modelling studies suggest that these biophysical components may be comparable with biochemical effects. In regard to climate change, the effects of these two distinct processes may counterbalance one another both regionally and, possibly, globally. In this article, through hypothetical large-scale deforestation simulations using a global climate model, we contrast the implications of afforestation on ameliorating or enhancing anthropogenic contributions from previously converted (agricultural) land surfaces. Based on our review of past studies on this subject, we conclude that the sum of both biophysical and biochemical effects should be assessed when large-scale afforestation is used for countering global warming, and the net effect on global mean temperature change depends on the location of deforestation/afforestation. Further, although biochemical effects trigger global climate change, biophysical effects often cause strong local and regional climate change. The implication of the biophysical effects for adaptation and mitigation of climate change in agriculture and agroforestry sectors is discussed. center dot Land-use changes affect global and regional climates through both biochemical and biophysical process. center dot Climate effect from biophysical process depends on the location of land-use change. center dot Climate mitigation strategies such as afforestation/reforestation should consider the net effect of biochemical and biophysical processes for effective mitigation. center dot Climate-smart agriculture could use bio-geoengineering techniques that consider plant biophysical characteristics such as reflectivity and water use efficiency.

Effects of self energy of the ions on the double layer structure and properties at the dielectric interface

Although numerous theoretical efforts have been put forth, a systematic, unified and predictive theoretical framework that is able to capture all the essential physics of the interfacial behaviors of ions, such as the Hofmeister series effect, Jones-Ray effect and the salt effect on the bubble coalescence remain an outstanding challenge. The most common approach to treating electrostatic interactions in the presence of salt ions is the Poisson-Boltzmann (PB) theory. However, there are many systems for which the PB theory fails to offer even a qualitative explanation of the behavior, especially for ions distributed in the vicinity of an interface with dielectric contrast between the two media (like the water-vapor/oil interface). A key factor missing in the PB theory is the self energy of the ion.

In this thesis, we develop a self-consistent theory that treats the electrostatic self energy (including both the short-range Born solvation energy and the long-range image charge interactions), the nonelectrostatic contribution of the self energy, the ion-ion correlation and the screening effect systematically in a single framework. By assuming a finite charge spread of the ion instead of using the point-charge model, the self energy obtained by our theory is free of the divergence problems and gives a continuous self energy across the interface. This continuous feature allows ions on the water side and the vapor/oil side of the interface to be treated in a unified framework. The theory involves a minimum set of parameters of the ion, such as the valency, radius, polarizability of the ions, and the dielectric constants of the medium, that are both intrinsic and readily available. The general theory is first applied to study the thermodynamic property of the bulk electrolyte solution, which shows good agreement with the experiment result for predicting the activity coefficient and osmotic coefficient.

Next, we address the effect of local Born solvation energy on the bulk thermodynamics and interfacial properties of electrolyte solution mixtures. We show that difference in the solvation energy between the cations and anions naturally gives rise to local charge separation near the interface, and a finite Galvani potential between two coexisting solutions. The miscibility of the mixture can either increases or decreases depending on the competition between the solvation energy and translation entropy of the ions. The interfacial tension shows a non-monotonic dependence on the salt concentration: it increases linearly with the salt concentration at higher concentrations, and decreases approximately as the square root of the salt concentration for dilute solutions, which is in agreement with the Jones-Ray effect observed in experiment.

Next, we investigate the image effects on the double layer structure and interfacial properties near a single charged plate. We show that the image charge repulsion creates a depletion boundary layer that cannot be captured by a regular perturbation approach. The correct weak-coupling theory must include the self-energy of the ion due to the image charge interaction. The image force qualitatively alters the double layer structure and properties, and gives rise to many non-PB effects, such as nonmonotonic dependence of the surface energy on concentration and charge inversion. The image charge effect is then studied for electrolyte solutions between two plates. For two neutral plates, we show that depletion of the salt ions by the image charge repulsion results in short-range attractive and long-range repulsive forces. If cations and anions are of different valency, the asymmetric depletion leads to the formation of an induced electrical double layer. For two charged plates, the competition between the surface charge and the image charge effect can give rise to like- charge attraction.

Then, we study the inhomogeneous screening effect near the dielectric interface due to the anisotropic and nonuniform ion distribution. We show that the double layer structure and interfacial properties is drastically affected by the inhomogeneous screening if the bulk Debye screening length is comparable or smaller than the Bjerrum length. The width of the depletion layer is characterized by the Bjerrum length, independent of the salt concentration. We predict that the negative adsorption of ions at the interface increases linearly with the salt concentration, which cannot be captured by either the bulk screening approximation or the WKB approximation. For asymmetric salt, the inhomogeneous screening enhances the charge separation in the induced double layer and significantly increases the value of the surface potential.

Finally, to account for the ion specificity, we study the self energy of a single ion across the dielectric interface. The ion is considered to be polarizable: its charge distribution can be self-adjusted to the local dielectric environment to minimize the self energy. Using intrinsic parameters of the ions, such as the valency, radius, and polarizability, we predict the specific ion effect on the interfacial affinity of halogen anions at the water/air interface, and the strong adsorption of hydrophobic ions at the water/oil interface, in agreement with experiments and atomistic simulations.

The theory developed in this work represents the most systematic theoretical technique for weak-coupling electrolytes. We expect the theory to be more useful for studying a wide range of structural and dynamic properties in physicochemical, colloidal, soft-matter and biophysical systems.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.

alpha-Tocopherol's Antioxidant Role: A Biophysical Perspective

I present evidence of an antioxidant mechanism for vitamin E that correlates strongly with its physical location in a model lipid bilayer. These data address the overlooked problem of the physical distance between the vitamin's reducing hydrogen and lipid acyl chain radicals. The combined data from neutron diffraction, NMR and UV spectroscopy experiments, all suggest that reduction of reactive oxygen species and lipid radicals occurs specifically at the membrane's hydrophobic-hydrophilic interface. The latter is possible when the acyl chain adopts conformations in which they snorkel to the interface from the hydrocarbon matrix. Moreover, not all model lipids are equal in this regard, as indicated by the small differences in the vitamin's location. The present result is a clear example of the importance of lipid diversity in controlling the dynamic structural properties of biological membranes. Importantly, these results suggest that measurements of alpha-tocopherol oxidation kinetics, and its products, should be revisited by taking into consideration the physical properties of the membrane in which the vitamin resides.

Plant roots release phospholipid surfactants that modify the physical and chemical properties of soil

Plant root mucilages contain powerful surfactants that will alter the interaction of soil solids with water and ions, and the rates of microbial processes. The lipid composition of maize, lupin and wheat root mucilages was analysed by thin layer chromatography and gas chromatography-mass spectrometry. A commercially available phosphatidylcholine (lecithin), chemically similar to the phospholipid surfactants identified in the mucilages, was then used to evaluate its effects on selected soil properties. The lipids found in the mucilages were principally phosphatidylcholines, composed mainly of saturated fatty acids, in contrast to the lipids extracted from root tissues. In soil at low tension, lecithin reduced the water content at any particular tension by as much as 10 and 50% in soil and acid-washed sand, respectively. Lecithin decreased the amount of phosphate adsorption in soil and increased the phosphate concentration in solution by 10%. The surfactant also reduced net rates of ammonium consumption and nitrate production in soil. These experiments provide the first evidence we are aware of that plant-released surfactants will significantly modify the biophysical environment of the rhizosphere.

Modification of cell wall properties in lettuce improves shelf life

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His(600). In the present work, the role of His(600) of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S(1) and S(1)`, specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His(600) residue makes important interactions with the substrate, supporting the prediction that His(600) moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K(m) and k(cat), showing the importance of His(600) for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His(600) in TOP catalysis, transferring a proton to the newly generated NH(2)-terminus or helping Tyr(605) and/or Tyr(612) in the intermediate oxyanion stabilization. (C) 2010 Elsevier Inc. All rights reserved.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PE and PS lipids synergistically enhance membrane poration by a peptide with anticancer properties

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.