Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide.
Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting that binding takes place at some distance from the plane of the polar groups. (31)P NMR indicates that the lipid bilayer remains intact upon R(9) binding. The fact that R(9) binds with greater affinity to HS than to anionic lipid vesicles makes the former molecule a more likely target in binding this CPP to the cell surface.
Gonçalves
Elisabete
E
Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Kitas
Eric
E
Seelig
Joachim
J
D000818:Animals; D000838:Anions / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002463:Cell Membrane Permeability; D041961:Deuterium Exchange Measurement; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry* / Q000378:metabolism; D010759:Phosphorus Isotopes / Q000378:metabolism; D011485:Protein Binding; D012542:Scattering, Radiation; D055672:Static Electricity; D013552:Swine; D013816:Thermodynamics
D000838:Anions; D001120:Arginine; D006497:Heparan Sulfate; D008051:Lipid Bilayers; D009842:Oligopeptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010759:Phosphorus Isotopes; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine