Protein Transduction Domains of HIV-1 and SIV TAT Interact with Charged Lipid Vesicles. Binding Mechanism and Thermodynamic Analysis
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- 作者：André ; Ziegler ; Xiaochun Li Blatter ; Anna Seelig ; and Joachim Seelig
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：August 5, 2003
- 年：2003
- 卷：42
- 期：30
- 页码：9185 - 9194
- 全文大小：114K
- 年卷期：v.42,no.30(August 5, 2003)
- ISSN：1520-4995
文摘
Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently bya mechanism not yet identified. Recent theories for the translocation suggest either the binding of theCPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively chargedlipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) andsimian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge zp =+8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized.Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires looselypacked monolayer films. For densely packed monolayers (
> 29 mN/m) and lipid bilayers, no insertionis possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scatteringexperiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralizedby TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane bindingwas quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is
H
-1.5 kcal/mol peptide and is almost temperature-independent with 
~0 kcal/(mol K),indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. Thebinding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partitionmodel. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for ~80%of the binding energy. The overall binding constant, Kapp, is ~103-104 M-1. The intrinsic binding constant(Kp), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is Kp ~1-10 M-1. Deuterium and phosphorus-31 nuclearmagnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMRdata provide no evidence for nonbilayer structures and also not for domain formation. This is furthersupported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interactionbetween TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroupconformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTDand the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranesthan for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding tobiological membranes.
> 29 mN/m) and lipid bilayers, no insertionis possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scatteringexperiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralizedby TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane bindingwas quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy isH -1.5 kcal/mol peptide and is almost temperature-independent with ~0 kcal/(mol K),indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. Thebinding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partitionmodel. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for ~80%of the binding energy. The overall binding constant, Kapp, is ~103-104 M-1. The intrinsic binding constant(Kp), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is Kp ~1-10 M-1. Deuterium and phosphorus-31 nuclearmagnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMRdata provide no evidence for nonbilayer structures and also not for domain formation. This is furthersupported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interactionbetween TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroupconformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTDand the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranesthan for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding tobiological membranes.