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Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47–60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 ± 0.6 μM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 ± 150 to 130 ± 30 μM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane. [ABSTRACT FROM AUTHOR]

Cyt1A is a cytolytic toxin produced by the spore-forming bacterium Bacillus thuringiensis var. israelensis, used in insecticide preparations. Understanding the molecular details of how the toxin changes conformation in the presence of lipid membrane is important for elucidating the toxin's mode of action. Previous binding studies were performed using membranes made of chemically undefined lipid egg phosphatidylcholine (PC). Here we studied lipid specificity of Cyt1A binding regarding saturation of fatty acyl chain and chemical nature of the lipid head-group. Fluorescence of tryptophan was used as a measure of binding. We measured Cyt1A binding to membranes made of the saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at 7°C and 37°C and determined the apparent dissociation constant (Kd) to be 111 ± 211 µM and 19.7 ± 5.9 µM, respectively. The apparent Kd of Cyt1A binding to membranes made of the egg PC lipid mixture was previously determined to be 91 µM. Thus at lower temperature, Cyt1A binds to membranes made of DMPC to a similar extent as to those made of the egg PC lipid mixture, but binds more strongly to DMPC at higher temperature. This suggests that Cyt1A binds to vesicles composed of either saturated or unsaturated lipids, and it binds saturated lipids in the liquid-crystalline state more strongly than unsaturated lipids. [ABSTRACT FROM AUTHOR]

The mechanism of internalization of cationic peptides into the cells is unknown. We used fluorescence spectroscopy to study the interaction between Protein Transduction Domain of the HIV-1 TAT protein (TAT-PTD; residues 47-60 of TAT, fluorescently labeled with tryptophan) and the lipid bilayer in the form of small unilamellar lipid vesicles (SUV). The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and increase in anisotropy upon the interaction with SUV, which was proportional with the negative charge density in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. Kd1 was independent of the negative charge density and accounted for 24% of the interaction, whereas Kd2, contributing 76% to the interaction, decreased linearly with the density of negative charge in the membrane, suggesting an electrostatic nature of the latter interaction. The former could not be inhibited by high salt, suggesting its van der Waals or hydrophobic nature. TAT-PTD did not dissipate membrane potential (165 mV, negative inside), nor did it affect fluorescence anisotropy of the membrane fluorescence probes TMA-DPH or DPH, indicating preserved membrane integrity upon TAT-PTD binding. When the pyrene-labeled phosphatidyl glycerol was included in the membrane, TAT-PTD induced pyrene excimer formation, but only at the temperature above the melting point of the lipid. Thus, the two prerequisites for a strong binding of TAT-PTD to the lipid membrane are the presence of the negative charge and the liquid crystalline phase of the lipid bilayer. [ABSTRACT FROM AUTHOR]

Abstract: The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide–lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley–White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence. [Copyright &y& Elsevier]