Interaction of the Effector Domain of MARCKS and MARCKS-Related Protein with Lipid Membranes Revealed by Electric Potential Measurements

Abstract

We have investigated the binding of the effector domains of myristoylated alanine-rich C kinase substrate (MARCKS) and of MARCKS-related protein (MRP) to lipid model membranes. For membrane systems we used lipid monolayers on a Langmuir trough and black lipid membranes (BLM). The binding of the peptides was detected by monitoring changes in the boundary potential of the lipid membranes. The vibrating plate technique (VPT) and the method of inner field compensation (IFC) were used for the monolayer and for the BLM, respectively. We could show that the effector domain of MARCKS binds to acidic lipid membranes mainly via electrostatic interactions and to zwitterionic lipid membranes via hydrophobic interactions. Isobaric measurements on lipid monolayers revealed that binding of both effector domains is accompanied by partial insertion of the peptides into the membrane. Adsorption and insertion of the peptides could be followed simultaneously by the VPT and by recording the increase in area of the lipid monolayer, respectively. No temporal delay could be observed between adsorption and insertion of the peptides, demonstrating that adsorption is the rate-limiting step and that insertion is faster than the time resolution of the experiments, i.e., a few seconds. Both the IFC and the VPT did not show any significant difference between the behaviors of the effector domains of MARCKS and MRP. With the IFC we show that calcium can regulate the translocation of the MARCKS effector peptide between the membrane and calmodulin (CaM) in the bulk. Our results indicate, that the IFC and VPT are suitable qualitatively, and to a certain extent quantitatively, as membrane binding assays.

PKC‐α controls MYD88‐dependent TLR/IL‐1R signaling and cytokine production in mouse and human dendritic cells

“…This is furthermore in line with the simultaneous timing and phoshorylation of MARCKS, an established PKC target also at the cell membrane. It is worth mentioning that phosphorylation of MARCKS by PKC leads to their detachment from the plasma membrane, which unmasks and increases the accessible lipid second messenger, PIP 2 , and is important for the activation of downstream signaling events [35][36][37]. Thus, our findings suggest that cPKC may play a more global role in TLR2-induced cellular processes and control phagocytosis, cytoskeletal changes and other signaling events.…”

Interactions of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS)-Related Protein with a Novel Solid-Supported Lipid Membrane System (TRANSIL)

“…The partition coefficients obtained by fitting the data to Eq. [5] (straight lines) as well as the standard deviation and the number of experiments can be found in Table 1 0.8 (22,29). We found that this method does not significantly change the estimation of K p (not shown).…”

“…K p was finally determined by fitting the resulting values of f to Eq. [5]. Note that it was not necessary to correct the data for the exclusion volume of the beads (e.g., 6% of the total volume at a total lipid concentration of 1 mM).…”

“…This signal can usually be followed by monitoring the spectroscopic properties of the membrane active substance, including fluorescence, circular dichroism, and infrared spectroscopy (2). Other methods focus on other intrinsic properties of the system, including heat changes, i.e., microcalorimetry (3) and potential changes induced at the surface of the membrane, e.g., inner field compensation (4,5) or the vibrating plate technique (5,6). Finally, spectroscopic techniques such as surface plasmon resonance (7,8) or two-mode waveguide spectroscopy (9,10) have been recently adapted to the field of membrane biology to allow an observation of the interaction of membraneactive molecules with bilayers in real time.…”

pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

“…This PD depends on many factors, such as the concentration of the electrolyte, its ability to penetrate the protein layer, and the distribution of fixed charges in this layer (35). Although the theory of the IFC method was developed to analyze the adsorption of small molecules, reliable results are obtained from the adsorption of large protein molecules as well (36)(37)(38). The simplest classic model of the relationship between the potential and surface charge density, which is proportional to the amount of protein molecules on the surface, is the equation of Gouy and Chapman (39,40),…”