Interaction of the effector domain of MARCKS and MARCKS-related protein with lipid membranes revealed by electric potential measurements.
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.
Bähr
G
G
Department of Biophysical Chemistry, Biozentrum of the University of Basel, Switzerland.
Diederich
A
A
Vergères
G
G
Winterhalter
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004553:Electric Conductivity; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C