Export of proteins across membranes: The helix reversion hypothesis

A model is presented which explains the biological role of the leader peptide in protein export. Along the lines of this model, the conformational changes of a protein with environment serves as a general mechanism for translocation. The leader peptide in the cytoplasm takes a hairpin like conformation which reverts to an extended helix upon integration into the membrane. The essential features of this model are in accord with recent results of protein export.

New computational approaches for wrinkled and slack membranes

The static response of thin, wrinkled membranes is studied using both a tension field approximation based on plane stress conditions and a 3D nonlinear elasticityformulation, discretized through 8-noded Cosserat point elements. While the tension field approach only obtains the wrinkled/slack regions and at best a measure of the extent of wrinkliness, the 3D elasticity solution provides, in principle, the deformed shape of a wrinkled/slack membrane. However, since membranes barely resist compression, the discretized and linearized system equations via both the approaches are ill-conditioned and solutions could thus be sensitive to discretizations errors as well as other sources of noises/imperfections. We propose a regularized, pseudo-dynamical recursion scheme that provides a sequence of updates, which are almost insensitive to theregularizing term as well as the time step size used for integrating the pseudo-dynamical form. This is borne out through several numerical examples wherein the relative performance of the proposed recursion scheme vis-a-vis a regularized Newton strategy is compared. The pseudo-time marching strategy, when implemented using 3D Cosserat point elements, also provides a computationally cheaper, numerically accurate and simpler alternative to that using geometrically exact shell theories for computing large deformations of membranes in the presence of wrinkles. (C) 2010 Elsevier Ltd. All rights reserved.

On the admittance of lipid bilayer membranes: Part I. Membrane-permeable ions

The modular formalism of Rangarajan [J. Electroanal. Chem., 55 (1974) 297] has been applied to the admittance of lipid bilayer membranes. The method leads to equations which clearly show the interrelations between the various partial processes involved in ion transport, and which allow examination of model assumptions without the need for a complete rederivation of the membrane admittance. Explicit expressions are given for both the continuum and single jump models. The former includes the ionic displacement component, important mostly at high frequencies.

On the admittance of lipid bilayer membranes: Part I. Membrane-permeable ions

The modular formalism of Rangarajan [J. Electroanal. Chem., 55 (1974) 297] has been applied to the admittance of lipid bilayer membranes. The method leads to equations which clearly show the interrelations between the various partial processes involved in ion transport, and which allow examination of model assumptions without the need for a complete rederivation of the membrane admittance. Explicit expressions are given for both the continuum and single jump models. The former includes the ionic displacement component, important mostly at high frequencies.

Sodium-alginate-based proton-exchange membranes as electrolytes for DMFCs

Novel mixed-matrix membranes prepared by blending sodium alginate (NaAlg) with polyvinyl alcohol (PVA) and certain heteropolyacids (HPAs), such as phosphomolybdic acid (PMoA), phosphotungstic acid (PWA) and silicotungstic acid (SWA), followed by ex-situ cross-linking with glutaraldehyde (GA) to achieve the desired mechanical and chemical stability, are reported for use as electrolytes in direct methanol fuel cells (DMFCs). NaAlg-PVA-HPA mixed matrices possess a polymeric network with micro-domains that restrict methanol cross-over. The mixed-matrix membranes are characterised for their mechanical and thermal properties. Methanol cross-over rates across NaAlg-PVA and NaAlg-PVA-HPA mixed-matrix membranes are studied by measuring the mass balance of methanol using a density meter. The DMFC using NaAlg-PVA-SWA exhibits a peak power-density of 68 mW cm(-2) at a load current-density of 225 mA cm(-2), while operating at 343 K. The rheological properties of NaAlg and NaAlg-PVA-SWA viscous solutions are studied and their behaviour validated by a non-Newtonian power-law.

A self-assembled, luminescent europium cholate hydrogel: a novel approach towards lanthanide sensitization

We propose a new self-assembly based strategy for the design of novel lanthanide based luminescent materials. In this approach a europium hydrogel is prepared and sensitization is achieved by doping the gel with pyrene in a non-coordinated fashion.

Generation of Flat and Curved Membranes by Inverse-BAR Domain Proteins

Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.

Lipid-amphotericin B complex structure in solution: A possible first step in the aggregation process in cell membranes

The interactions between the polyene antibiotic amphotericin B with dipalmitoylphosphatidylcholine were investigated in vesicles (using circular dichroism) and in chloroform solution (using circular dichroism and IH, I3C, and 31P nuclear magnetic resonance). The results show that amphotericin B readily aggregates in vesicles and that the extent of aggregation depends on the 1ipid:drug concentration ratio. Introduction of sterol molecules into the membrane hastens the process of aggregation of amphotericin B. In chloroform solutions amphotericin B strongly interacts with phospholipid molecules to form a stoichiometric complex. The results suggest that there are interactions between the conjugated heptene stretch of amphotericin B and the methylene groups of lipid acyl chains, while the sugar moiety interacts with the phosphate head group by the formation of a hydrogen bond. A model is proposed for the lipid-amphotericin B complex, in which amphotericin B interacts equally well with the two lipid acyl chains, forming a 1:l complex.

Ion probing with Luminescent PbS Quantum dots in PVA

The PbS quantum dots synthesized in PVA are used to investigate their photoluminescence (PL) response to various ions such as Zn, Cd, Hg, Ag, Cu, Fe, Mn, Co, Cr and Ni ions. The enhancement in the photoluminescence intensity is observed with specific ions namely Zn, Cd, Hg and Ag. Among these four ions, the PL response to Hg and Ag even at sub-micro-molar concentrations is quite high, approximately an order of magnitude higher than Zn and Cd. It is interesting to observe that the change in Pb and S molar ratio has profound effect on the selectivity of these ions. The samples prepared under excess of S are quite effective compared to Pb. Indeed, the later one has hardly any effect on the photoluminescence response. These results also indicate that the sensitivity of these QDs could be fine-tuned by controlling the S concentration at the surface. Contrary to the above, Cu, Fe and Co quenches the photoluminescence. Another interesting property of PbS in PVA observed is photo-brightening mechanism due to the curing of the polymer with laser. However, the presence of excess ions at the surface changes its property to photo-darkening/brightening that depends on the direction of carrier transfer mechanism (from QDs to the surface adsorbed metal ions or vice-versa), which is an interesting feature for metal ion detectivity.

Membranes of Cationic Gemini Lipids based on Cholesterol with Hydroxyl Headgroups and their Interactions with DNA and Phospholipid

Two series of cholesterol-based cationic gemini lipids with and without hydroxyl functions at the headgroups possessing different lengths of polymethylene -(CH2)(n)-] (n = 3, 4, 5, 6, 12) spacer have been synthesized. Each gemini lipid formed stable suspension in water. The suspensions of these gemini lipids in water were investigated using transmission electron microscopy, dynamic light scattering, zeta potential measurements and X-ray diffraction to characterize the nature of the individual aggregates formed therein. The aggregation properties of these gemini lipids in water were found to strongly depend upon the length of the spacer and the presence of hydroxyl group at the headgroup region. Lipoplex formation (DNA binding) and the release of the DNA from such lipoplexes were performed to understand the nature of interactions that prevail between these cationic cholesterol aggregates and duplex DNA. The interactions between such gemini lipids and DNA depend both on the presence of OH on the headgroups and the spacer length between the headgroups. Finally, we studied the effect of incorporation of each cationic gemini lipid into dipalmitoyl phosphatidylcholine vesicles using differential scanning calorimetry. The properties of the resulting mixed membranes were found again to depend upon the nature of the headgroup and the spacer chain length.

Lanthanide Luminescent Coordination Polymer Constructed from Unsymmetrical Dinuclear Building Blocks Based on 4-((1H-Benzod]imidazol-1-yl)methyl)benzoic Acid

Lanthanide coordination polymers of the general formula Ln(2)(L)(5)(NO3)(H2O)(4)](n) (Ln = Eu (1), Tb (2), Gd (3)) supported by a novel aromatic carboxylate ligand 4-((1H-benzod]imidazol-1-yl)methyl)benzoic acid (HL) have been synthesized, characterized, and their photoluminescence behavior is examined. The powder X-ray diffraction patterns of complexes 1-3 showed that 1-3 are isostructural; thus, 1 has been chosen as an example to discuss in detail about the molecular structure by single-crystal X-ray diffraction. Complex 1 is a one-dimensional (1D) helical chain-like coordination polymer consisting of unique unsymmetrical dinuclear lanthanide building blocks. The 1D chains are further linked by the significant intermolecular hydrogen-bonding interactions to form a two-dimensional supramolecular network. The Tb3+ complex exhibits bright green luminescence efficiency in the solid state with a quantum yield of 15%. On the other hand, poor luminescence efficiency has been noted for Eu3+-benzoate complex.

Diacylglycerol Kinase Is Stimulated by Arachidonic Acid in Neural Membranes.

The effect of arachidonic acid (AA) on the activity of diacylglycerol (DG) kinase in neural membranes was investigated. When rat brain cortical membranes were incubated with 0.5 mM dipalmitin and [gamma-P-32]ATP, formation of phosphatidic acid (PA) was observed. It was linear up to 5 min, and the initial rate was similar to 1.0 nmol/min/mg of protein. The DG kinase activity was stimulated twofold by 0.25 mM AA. The stimulation was apparent at the earliest time point measured (1 min) and with the lowest concentration of AA tested (62.5 mu M). The stimulation was proportional to the concentration of AA up to 250 mu M. AA was the most potent stimulator of DG kinase, and linolenic acid showed similar to 40% stimulation. Oleic acid showed no effect, whereas linoleic and the saturated fatty acids tested were inhibitory. AA stimulation of DG kinase was observed only with membranes of cerebrum, cerebellum, and myelin and not with brain cytosol or liver membranes. AA also stimulated the formation of PA in the absence of added dipalmitin (endogenous activity) with membranes prepared from whole brain. DG kinase of neural membranes was extracted with 2 M NaCl, which on dialysis yielded a precipitate. Both the precipitate and the supernatant showed DG kinase activity, but only the enzyme in the precipitate was stimulated by AA at concentrations as low as 25 mu M. It is suggested that AA, through its effect on DG kinase, regulates the level of DG in neural membranes, which in turn regulates protein kinase C activity.

Regulation of diacylglycerol kinase in rat brain membranes by docosahexaenoic acid

The effect of docosahexaenoic acid (DHA) on the diacylglycerol kinase (DG kinase) activity in rat brain membranes was investigated. DHA at 500 mu M concentration, stimulated the enzyme activity by about 2 fold. This effect was concentration-and time-dependent and was observed after very short periods of incubation (one min). DHA stimulation of DG kinase was observed only with rat brain membranes, and not with rat brain cytosol or rat liver membranes. Treating the rat brain membranes with phospholipase A(2) which released free fatty acids including DHA, significantly stimulated the DG kinase activity. It is concluded that DHA through its stimulatory effect on DG kinase may regulate the signalling events in growth-related situations in the brain such as synaptogenesis.

Verifying scalings for bending rigidity of bilayer membranes using mesoscale models

The bending rigidity kappa of bilayer membranes was studied with coarse grained soft repulsive potentials using dissipative particle dynamics (DPD) simulations. Using a modified Andersen barostat to maintain the bilayers in a tensionless state, the bending rigidity was obtained from a Fourier analysis of the height fluctuations. From simulations carried out over a wide range of membrane thickness, the continuum scaling relation kappa proportional to d(2) was captured for both the L-alpha and L-beta phases. For membranes with 4 to 6 tail beads, the bending rigidity in the L-beta phase was found to be 10-15 times higher than that observed for the L-alpha phase. From the quadratic scalings obtained, a six fold increase in the area stretch modulus, k(A) was observed across the transition. The magnitude of increase in both kappa and k(A) from the L-alpha to the L-beta phase is consistent with current experimental observations in lipid bilayers and to our knowledge provides for the first time a direct evaluation of the mechanical properties in the L-beta phase.

Surfactant lipids containing aromatic units produce vesicular membranes with high thermal stability

Six new vesicle-forming, cationic surfactant lipids are synthesized. Four of them contain 'flat' aromatic units at different locations of hydrophobic segments. In order to estimate the influence of aromatic units in the lipid monomer two other surfactant lipids of related structure with n-butyloxy units in the places of aromatic groups were also prepared. Transmission electron microscopy confirmed the vesicular membrane formation from these newly synthesized lipids. DSC or temperature-dependent keto-enol tautomerism of benzoylacetanilide-doped vesicles reveal a remarkable increase in the thermal stability of the membranes formed from aromatic surfactant lipids in contradistinction to their counterparts that contain n-butyloxy units. The enhanced thermal stability originates presumably as a consequence of inter-monomer stacking.