Sanat Karmakar

Binding of monovalent alkali metal ions with negatively charged phospholipid membranes.

We have systematically investigated the effect of various alkali metal ions with negatively charged phospholipid membranes. Size distributions of large unilamellar vesicles have been confirmed using dynamic light scattering. Zeta potential and effective charges per vesicle in the presence of various alkali metal ions have been estimated from the measured electrophoretic mobility. We have determined the intrinsic binding constant from the zeta potential using electrostatic double layer theory. The reasonable and consistent value of the intrinsic binding constant of Na(+), found at moderate NaCl concentration (10-100 mM), indicates that the Gouy-Chapman theory cannot be applied for very high (> 100mM) and very low (< 10 mM) electrolyte concentrations. The isothermal titration calorimetry study has revealed that the net binding heat of interaction of the negatively charged vesicles with monovalent alkali metal ions is small and comparable to those obtained from neutral phosphatidylcholine vesicles. The overall endothermic response of binding heat suggests that interaction is primarily entropy driven. The entropy gain might arise due to the release of water molecules from the hydration layer vicinity of the membranes. Therefore, the partition model which does not include the electrostatic contribution suffices to describe the interaction. The binding constant of Na(+) (2.4 ± 0.1 M(-1)), obtained from the ITC, is in agreement with that estimated from the zeta potential (-2.0 M(-1)) at moderate salt concentrations. Our results suggest that hydration dynamics may play a vital role in the membrane solution interface which strongly affects the ion-membrane interaction.

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Unstable crack propagation in LAPONITE® gels: selection of a sinusoidal mode in an electric field

We report observation of wavy cracks and naturally patterned fracture surfaces in drying LAPONITE® paste. Desiccation cracks are shown to follow undulating, corrugated paths even when the speed of crack propagation is lower than the sound velocity in the medium by two orders of magnitude. Fast Fourier transform of the wavy crack path shows that it is a superposition of several sinusoidal modes and their harmonics. When the paste is exposed to a DC electric field during drying, by imposing a 50 V potential, some of the modes are suppressed. Increasing the voltage to 100 V results in survival of only one pure sinusoidal mode of wavelength ∼292 μm. We suggest that an effective mixed mode loading develops as a result of faster evaporation at the upper surface of the paste, and this is responsible for the instability leading to the wavy contour of the crack. The present study provides an insight into the mechanism of wavelength selection under an electric field of sufficient strength. We also show that unstable crack propagation may have similarity with the mechanism that exists in an auxiliary experiment: breaking of a perspex sheet.

Pattern formation of drying lyotropic liquid crystalline droplet

We present a study of pattern formation in drying sessile droplets of aqueous solutions of cetyltrimethylammonium bromide (CTAB)–water system using polarising optical microscopy (POM) and computer simulation. A 4-fold symmetrical and a 3 dimensional staircase like pattern appear during drying of the droplets of the CTAB–water system. However, when NaBr salt is added to the solution, the pattern changes to a system of concentric rings with a fainter set of hyperbolic fringes superimposed on it. The ring width is found to decrease from the central to the peripheral region. We compared the CTAB–water–NaBr pattern with patterns obtained by simulating a 3-dimensional model with various director configurations using a Jones matrix calculation. A good qualitative agreement of the simulated and experimental patterns is obtained for a model assuming a lenticular shaped droplet with the local directors parallel to each other and aligned horizontally, parallel to the fluid–substrate interface. The circular and hyperbolic fringe patterns as well as variation in width of rings are qualitatively reproduced by the simulation. When the droplet dries out completely dendritic crystals form for the CTAB–water droplet as well as the CTAB–water–NaBr droplet. Our results indicate that as the droplet concentration increases with evaporation, elongated cylindrical micelles form which prefer a mutually parallel, horizontal configuration.

Chapter Seven - X-ray and Neutron Scattering Studies of Lipid–Sterol Model Membranes

Abstract Sterols are major components of many biomembranes and are known to play an important role in several biological processes. In order to understand the complex lipid–sterol interactions and their influence on membrane structure and properties, model membranes containing cholesterol and other sterols have been widely studied using a variety of experimental techniques. This chapter gives a brief review of X-ray and neutron scattering studies of these systems, highlighting the detailed structural information they provide.

Investigation of ionic conduction in PEO–PVDF based blend polymer electrolytes

We investigate the effect of blend host polymer on solid polymer electrolyte (SPE) films doped with ammonium iodide (NH4I) salt using a variety of experimental techniques. Structural studies on the composite SPEs show that the blending of Poly(ethylene oxide) (PEO)–Poly(vinylidene fluoride) (PVDF) polymers in a suitable ratio enhances the amorphous fraction of the polymer matrix and facilitates fast ion conduction through it. We observe that the addition of a small amount of PVDF in the PEO host polymer enhances the ion – polymer interaction leading to more ion dissociation. As a result, the effective number of mobile charge carriers within the polymer matrix increases. Systematic investigation in these blend SPEs shows that the maximum conductivity (1.01 × 10–3 S/cm) is obtained for PEO – rich (80 wt. % PEO, 20 wt. % PVDF) composites at 35 wt. % NH4I concentration at room temperature. Interestingly, at higher salt concentrations (above 35 wt. %), the conductivity is found to decrease in this system. The reduction of conductivity at higher salt concentrations is the consequence of decrease in the carrier concentration due to the formation of an ion pair and ion aggregates. PVDF–rich compositions (20 wt. % PEO and 80 wt. % PVDF), on the other hand, show a very complex porous microstructure. We also observe a much lower ionic conductivity (maximum ∼ 10–6 S/cm at 15 wt. % salt) in these composite systems relative to PEO-rich composites.We investigate the effect of blend host polymer on solid polymer electrolyte (SPE) films doped with ammonium iodide (NH4I) salt using a variety of experimental techniques. Structural studies on the composite SPEs show that the blending of Poly(ethylene oxide) (PEO)–Poly(vinylidene fluoride) (PVDF) polymers in a suitable ratio enhances the amorphous fraction of the polymer matrix and facilitates fast ion conduction through it. We observe that the addition of a small amount of PVDF in the PEO host polymer enhances the ion – polymer interaction leading to more ion dissociation. As a result, the effective number of mobile charge carriers within the polymer matrix increases. Systematic investigation in these blend SPEs shows that the maximum conductivity (1.01 × 10–3 S/cm) is obtained for PEO – rich (80 wt. % PEO, 20 wt. % PVDF) composites at 35 wt. % NH4I concentration at room temperature. Interestingly, at higher salt concentrations (above 35 wt. %), the conductivity is found to decrease in this system. The ...

Effect of counterions on the binding affinity of Na+ ions with phospholipid membranes

We have systematically investigated the effect of counterions on the interaction of the Na+ ion with phospholipid membranes using dynamic light scattering, zeta potential, isothermal titration calorimetry and fluorescence spectroscopy techniques. As model membranes, large unilamellar vesicles (LUV) have been prepared using an extrusion method and their size distribution confirmed using dynamic light scattering. The zeta potential of LUV in the presence of different sodium salts has been estimated from the measured electrophoretic mobility. The intrinsic binding constant of Na+ in the presence of various counterions, such as Cl−, Br−, and I− was derived from the zeta potential using Gouy–Chapman theory at moderate salt concentrations (10–100 mM). The apparent binding constant estimated from ITC is in agreement with that obtained from the zeta potential. The overall endothermic response of binding heat suggests that the ion–membrane interaction is primarily entropy driven. The entropy gain might arise due to release of water molecules from the hydration layer vicinity of the membranes. The effect of counterions on the binding affinity of Na+ follows the order I− > Br− > Cl− for neutral DOPC membranes. However, for negatively charged membranes, the order is Br− > I− > Cl−. The results of fluorescence spectroscopy also compliment the zeta potential and ITC results. Fluorescence lifetime and anisotropy experiments suggest that among all anions I− adsorbs and penetrates into the membrane, indicating a significant effect of the I− ion compared to other anions. Our result is in agreement with the earlier simulation study on this system.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by ~10nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Crack Patterns in Drying Laponite–NaCl Suspension: Role of the Substrate and a Static Electric Field

We report the formation of crack patterns in drying films of Laponite-NaCl solution. Crack patterns that develop upon drying aqueous Laponite-NaCl solution change drastically as the amount of NaCl is varied in the solution. In this work, we have investigated the effect of NaCl on drying films of aqueous solution of Laponite under two conditions: (i) when the film is bounded by a wall, as in Petri dish experiments and (ii) when the film does not have any boundary, as in experiments with droplets. In order to obtain insights into the effect of the substrate, the experiments have been done with two different substrates of different hydrophobicities, polypropylene and glass. The formation of crack patterns has been explained on the basis of the wetting and spreading properties of the solution on these substrates and the effect of salt on colloidal aggregation. In this work, we have shown that the presence of salt in aqueous Laponite solution can induce crack patterns depending on the nature of the substrate. Another important aspect of this work is the role of NaCl in crack inhibition in desiccating films of aqueous Laponite, in the presence of static electric field. This effect can be utilized to suppress undesirable crack formation in many applications.

Effect of Zwitterionic Phospholipid on the Interaction of Cationic Membranes with Monovalent Sodium Salts

Cationic lipids have attracted much attention because of their potential for biomedical applications, such as gene delivery. The gene transfection efficiency of cationic lipids is greatly influenced by the counterions as well as salt ions. We have systematically investigated the interaction of different monovalent sodium salts with positively charged membrane, composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP, using dynamic light scattering, zeta potential, isothermal titration calorimetry (ITC), and fluorescence spectroscopy techniques. Our results reveal that the affinity of anions with cationic membranes follows the sequence I- ≫ Br- > Cl- according to descending order of their sizes and is consistent with the Hofmeister series. Interestingly, the electrostatic behavior of the DOTAP membrane in the presence of monovalent anions differs significantly from the DOPC/DOTAP membrane. This difference is due to the strong interplay between phosphocholine and trimethylammonium-propane (TAP) headgroups leading to the reorientation of the TAP group in the membrane. The binding constant of anions, derived from zeta potential and ITC is in agreement with the affinity of anions mentioned above. Among all anions, I- shows strongest affinity, as evidenced from the rapid increase in hydrodynamic radius which eventually leads to the formation of large aggregates. The fluorescence spectroscopy of a lypophilic probe Nile red in the presence of cationic vesicles containing ions complements the I- adsorption onto the membrane. Nonlinear Stern-Volmer plot, consisting of accessible and inaccessible Nile red to I- is consistent with the zeta potential as well as ITC results.