THE INFLUENCE OF DOPC ON THE PERMEABILITY OF LIPID MEMBRANE: DETERGENT SOLUBILIZATION AND NMR STUDY

Abstract. There is an efficient, systemic transgene expression in many cell lines (in vitro) by using anionic liposomes, composed of equimolar amounts of 1, 2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and cholesterol (CHOL). 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is added to this liposomal formulation to increase the stability of small unilamellar vesicles in the presence of fetal calf serum (FCS). The liposomal stability was studied by monitoring the retention of a trapped marker, carboxyfluorescein (CF), in the internal liposome compartment. The liposomes containing DOPC had a lower stability in serum compared to the DOPC free liposomes. The extremely low retention in the DOPC liposome was ascribed to a formation of the inverted hexagonal phase of the liposomal membrane. A higher sodium deoxycholate detergent concentration is needed to solubilize DLPE/DOPS/CHOL (1:1:1) liposomes than DLPE/DOPC/DOPS/CHOL (1:1:1:1) liposomes. Phosphorus-31 nuclear magnetic resonance ( 31 P-NMR) is used to investigate the lamellar (L α ) → hexagonal (H II ) phase transition of unilamellar lipid vesicles containing DOPC. It was found that unilamellar lipid vesicles containing DOPC are in the bilayer state at pH 4 and undergo a lamellar (L α ) → hexagonal (H II ) phase transition at pH 8

3 Enthalpy-entropy compensation: the role of solvation

complete understanding of EEC be obtained. Faced with such large, and compensating, changes in the enthalpies and entropies of binding, the best approach to engineering elevated affinities must be through the addition of ionic links, as they generate increased entropy without affecting the enthalpy. Keywords Enthalpy-entropy compensation (EEC) · Solvation water · Hydration · Proteins · DNA Enthalpy-entropy compensation Efforts to establish structure-activity relationships (SARs) and improve the affinity of drugs for target proteins typically involve thermodynamic measurements on a panel of modified forms of the lead compound, principally using isothermal titration calorimetry (ITC). It is frequently observed that whereas the Gibbs energy of binding, i.e., the binding constant, remains largely unchanged in consequence of the addition/subtraction of chemical groups, there are substantial variations in the component enthalpies and entropies. If ∆G remains the same, it follows that changes in ∆H and T∆S compensate one another. In fact, enthalpy-entropy compensation (EEC) is a widely observed phenomenon and is typically explained by assuming that if a molecular change in the ligand leads to more and/or tighter van der Waals contacts and H-bonds with the substrate (giving a more negative ∆H), this inevitably leads to reduced mobility/flexibility in either or both components of the interaction, i.e., a reduction in the overall conformational entropy, and that change compensates the enthalpy decrease. However, the amount of water hydrating the system can also change and if any of this water is tightly bound, its contribution to the enthalpy and entropy of binding will also be Abstract Structural modifications to interacting systems frequently lead to changes in both the enthalpy (heat) and entropy of the process that compensate each other, so that the Gibbs free energy is little changed: a major barrier to the development of lead compounds in drug discovery. The conventional explanation for such enthalpy-entropy compensation (EEC) is that tighter contacts lead to a more negative enthalpy but increased molecular constraints, i.e., a compensating conformational entropy reduction. Changes in solvation can also contribute to EEC but this contribution is infrequently discussed. We review long-established and recent cases of EEC and conclude that the large fluctuations in enthalpy and entropy observed are too great to be a result of only conformational changes and must result, to a considerable degree, from variations in the amounts of water immobilized or released on forming complexes. Two systems exhibiting EEC show a correlation between calorimetric entropies and local mobilities, interpreted to mean conformational control of the binding entropy/free energy. However, a substantial contribution from solvation gives the same effect, as a consequence of a structural link between the amount of bound water and the protein flexibility. Only by assuming substantial changes in solvation-an intrinsically compensatory process-can a more * Colyn Crane-Robinso

VIPP1 rods engulf membranes containing phosphatidylinositol phosphates

In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes