Structure and position of the N-terminal membrane-binding domain of pp60src at the membrane interface.

Hydrophobic and electrostatic interactions between the acylated N-terminal end of Src and lipid bilayers are responsible for the attachment of this nonreceptor tyrosine kinase to the membrane-solution interface. To investigate the structure and dynamics of this domain at the membrane interface, a series of peptides based upon the N-terminal end of pp60src, myr-src(2-16), was synthesized with single-site cysteine substitutions and derivatized with a sulfhydryl-reactive proxyl nitroxide. The EPR line shapes and mobility of these peptides when bound to the membrane interface were consistent with an extended peptide conformation, and no evidence was found for either a helical or sheet structure. Line shapes on the myristoylated N-terminal end indicate that this segment is more restricted in its motion than at the C-terminus. Although the membrane affinity of this peptide is much stronger in the presence of acidic lipid, EPR line shapes were not strongly affected by the presence of acidic lipid. An EPR power saturation technique was used to provide information on the position of nitroxides from the interface for the membrane-bound peptide. When membrane bound, labeled side chains at the N-terminal end of the peptide were found to lie in the aqueous phase near the membrane interface; however, for the C-terminal half of the peptide, residues were further off the membrane and were 10-15 A from the interface. Peptides derived from the membrane and calmodulin binding domains of the myristoylated alanine-rich C kinase substrate and neuromodulin were previously found to be in extended conformations; however, side chains for these peptides penetrated the membrane-solution interface. We speculate that the relatively polar character of the N-terminal segment of Src and a Born repulsion energy prevent this peptide from penetrating into the membrane interface when membrane bound.