Membrane structure of protein kinase C and calmodulin binding domain of myristoylated alanine rich C kinase substrate determined by site-directed spin labeling.

Cysteine-substituted peptides based on the membrane, calmodulin, and protein kinase C binding domain of the myristoylated alanine rich C kinase substrate (MARCKS) were synthesized and derivatized with a sulfhydryl reactive proxyl nitroxide. These spin-labeled peptides were used in combination with continuous wave power saturation electron paramagnetic resonance (EPR) spectroscopy to determine the position and structure of the peptide on membranes containing phosphatidylserine. These peptides bind at the membrane interface, with nitroxide side chains in the central and C-terminal regions lying several angstroms below the level of the head group. In contrast, the N-terminus of the peptide is extended out of the membrane interface so that the two N-terminal residues are positioned on the aqueous side of the head group. When bound to the membrane, the N-terminal segment of this peptide is sensitive to the membrane surface charge density. Higher charge densities decrease the amplitude of side chain motions at the N-terminus and bring this end of the peptide closer to the membrane interface. When the location of successive residues along the bilayer normal is compared, no helical trend is seen, and no evidence for aggregation of the peptide is found. The EPR spectra of double spin-labeled peptides also show no evidence for a helical structure. Thus, these basic peptides are in an extended configuration at the membrane interface with hydrophobic side chains oriented inward toward the membrane hydrocarbon.