Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.
Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The noncharged species can easily cross the lipid membrane. We have measured with solid-state NMR the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of DeltapK(a) = 1.2 units in neutral lipid membranes and the percentage of the noncharged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine, and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of P-glycoprotein in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the P-glycoprotein active center if the reaction takes place exclusively in the lipid matrix.
Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/ m). S19 partitions into charged monolayers/bilayers with a penetration area A p ؍ 140 ؎ 30 Å 2 and a free energy of binding of ⌬G 0 ؍ ؊5.7 kcal/mol, thereby forming a partially ␣-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A p ؍ 148 ؎ 23 Å 2 and A p ؍ 160 ؎ 15 Å 2 , respectively, forming an ␣-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is ⌬G 0 ؍ ؊9.4 kcal/mol with a hydrophobic contribution of ⌬G h ؍ ؊7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of ⌬G h ؍ ؊1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.Talin is a widespread actin-binding protein present in focal cell adhesions and ruffling membranes of moving cells (1, 2). In fibroblasts, talin binding to lipid membranes is associated with the establishment of a signaling cascade, mediated either by integrins (3, 4) leading to the formation of focal adhesions or, alternatively, by layilin (5) leading to a nucleation of actin assembly in membrane ruffles. In platelets, talin redistributes from the cytoplasm to the membrane during activation (6) where it colocalizes with the GPIIb/IIIa complex (7). Talin analogues have been identified in lower organisms like Dictyostelium (8) and Caenorhabditis elegans (9), the N termini and C termini being most preserved.Reasons to assume that talin is involved in a polarized assembly of the actin cytoskeleton by nucleating actin filament growth at lipid interfaces (10, 11) were provided from the finding that Dictyostelium mutants, which lack the entire protein, are massively impaired in adhesion and motility (12), and HeLa cells, when down-regulated in talin expression by antisense RNA, exhibit a reduced rate in cell spreading (13). Antibodies directed against talin, when microinjected were shown to inhibit fibroblast migration (14).Some of talin's functions have been attributed to specific protein domains. Calpain or thrombin cleavage in vitro yields two parts with 190 and 47 kDa, respectively. The C-terminal 190-kDa portion was shown to carry the actin binding sites (15) in form of a conserved sequence, the (I/L)WEQ module (16), and to be responsible f...
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