Membrane Binding of Peptides Containing Both Basic and Aromatic Residues. Experimental Studies with Peptides Corresponding to the Scaffolding Region of Caveolin and the Effector Region of MARCKS

Abstract

We have studied the binding of peptides containing both basic and aromatic residues to phospholipid vesicles. The peptides caveolin(92-101) and MARCKS(151-175) both contain five aromatic residues, but have 3 and 13 positive charges, respectively. Our results show the aromatic residues insert into the bilayer and anchor the peptides weakly to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC). Incorporation of a monovalent acidic lipid (e.g., phosphatidylserine, PS) into the vesicles enhances the binding of both peptides via nonspecific electrostatic interactions. As predicted from application of the Poisson-Boltzmann equation to atomic models of the peptide and membranes, the enhancement is larger (e.g., 10(4)- vs 10-fold for 17% PS) for the more basic MARCKS(151-175). Replacing the five Phe with five Ala residues in MARCKS(151-175) decreases the binding to 10:1 PC/PS vesicles only slightly (6-fold). This result is also consistent with the predictions of our theoretical model: the loss of the attractive hydrophobic energy is partially compensated by a decrease in the repulsive Born/desolvation energy as the peptide moves away from the membrane surface. Incorporating multivalent phosphatidylinositol 4, 5-bisphosphate (PIP(2)) into PC vesicles produces dramatically different effects on the membrane binding of the two peptides: 1% PIP(2) enhances caveolin(92-101) binding only 3-fold, but increases MARCKS(151-175) binding 10(4)-fold. The strong interaction between the effector region of MARCKS and PIP(2) has interesting implications for the cellular function of MARCKS.

Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions

“…2A shows that FA-MARCKS (‚) binds 100-fold less strongly to 99:1 PC/PIP 2 vesicles than MARCKS-(151-175) (E). The observation that the 5 Phe residues contribute significantly (ϳ3 kcal/mol) to the binding energy is consistent with previous observations that the Phe residues of MARCKS-(151-175) penetrate the polar head group region of the membrane (40,45,72). The aromatic residues contribute to the binding of MARCKS-(151-175) not only to PC/PIP 2 vesicles but also to PC/PS vesicles.…”

“…There are several possible explanations for the less-than-a-maximal effect observed with our charged peptides. For example, a decrease in free energy due to penetration of aromatics into the bilayer is accompanied by an increase in Born/dehydration free energy required to move the charges closer to the interface, as discussed in detail elsewhere (33,45).…”

“…We labeled peptides with radioactive [ 3 H]NEM as described previously (45). Briefly, we placed 250 Ci of [ 3 H]NEM in pentane on top of 20 l of DMF, evaporated the pentane with argon gas, and then mixed the [ 3 H]NEM in DMF with 1 ml of an ϳ1 mM peptide solution; this TABLE I Sequences of peptides Basic residues Lys and Arg are in bold.…”

“…Its binding to PC/PIP 2 vesicles depends on a complicated interplay of at least two factors that drive adsorption, electrostatic attraction of basic residues for the surface and hydrophobic insertion of aromatics into the polar head group region, and two factors that oppose adsorption, Born/dehydration repulsion and the entropy price required to pull several PIP 2 lipids together. These factors (except the entropy price) have been discussed elsewhere (33,45). We investigated a simpler system, measuring the binding of peptides that contain only basic residues.…”

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

“…Twenty-two fragment ions matched, with no discrepancies. Fragment ion z 7 indicates phosphorylation on Ser 7 or Ser 10 whereas fragment ion c 10 indicates that any of the three serine residues may be modified.…”