Effect of cholesterol on the ion-membrane interaction: Zeta potential and dynamic light scattering study.

Cholesterol in a bio-membrane plays a significant role in many cellular event and is known to regulate the functional activity of protein and ion channel. In this study we report a significant effect of cholesterol on the ion-membrane interaction. We prepare large unilamellar vesicles, composed of zwitterionic lipid DOPC and anionic lipid DOPG with different cholesterol concentration. Electrostatics of anionic membranes containing cholesterol in the presence of NaCl has systematically been explored using dynamic light scattering and zeta potential. Negative zeta potential of the membrane decreases its negative value with increasing ion concentration for all cholesterol concentrations. However, zeta potential itself decreases with increasing cholesterol content even in the absence of monovalent ions. Electrostatic behaviour of the membrane is determined from well-known Gouy Chapmann model. Negative surface charge density of the membrane decreases with increasing cholesterol content. Binding constant, estimated from the electrostatic double layer theory, is found to increase significantly in the presence of cholesterol. Comparison of electrostatic parameters of the membrane in the presence and absence of cholesterol suggests that cholesterol significantly alter the electrostatic behaviour of the membrane.

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.

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.

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.

Charge-Driven Interaction of Antimicrobial Peptide NK-2 with Phospholipid Membranes.

NK-2, derived from a cationic core region of NK-lysin, displays antimicrobial activity toward negatively charged bacterial membranes. We have studied the interaction of NK-2 with various phospholipid membranes, using a variety of experimental techniques, such as, isothermal titration calorimetry (ITC), ζ potential, and dynamic light scattering. As bacteria mimicking membranes, we have chosen large unilamellar vesicles (LUVs) composed of negatively charged phospholipid and neutral phospholipids. ITC and ζ potential results show the stronger binding affinity of NK-2 to negatively charged membranes than to neutral membranes. Saturation of the isotherm, obtained from ITC, at a given lipid to NK-2 ratio, was found to be consistent with the charge compensation, determined from ζ potential. A surface partition model with electrostatic contribution was used to estimate the intrinsic binding constant and other thermodynamical parameters of binding kinetics of NK-2. The size distribution of negatively charged LUV in the presence of NK-2 was found to increase drastically, indicating the presence of large aggregates. Such a large aggregate has not been observed in neutral membranes, which supports the ITC and ζ potential results.

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.