STRUCTURE AND POSITION OF THE N-TERMINAL MEMBRANE-BINDING DOMAIN OF PP60(SRC) AT THE MEMBRANE INTERFACE

Hydrophobic and electrostatic interactions between the acylated N-term inal 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 memb rane interface, a series of peptides based upon the N-terminal end of pp60(src), myr-src(.2-16), was synthesized with single-site cysteine s ubstitutions and derivatized with a sulfhydryl-reactive proxyl nitroxi de. The EPR line shapes and mobility of these peptides when bound to t he membrane interface were consistent with an extended peptide conform ation, and no evidence was found for either a helical or sheet structu re. Line shapes on the myristoylated N-terminal end indicate that this segment is more restricted in its motion than at the C-terminus. Alth ough the membrane affinity of this peptide is much stronger in the pre sence of acidic lipid, EPR line shapes were not strongly affected by t he presence of acidic lipid. An EPR power saturation technique was use d to provide information on the position of nitroxides from the interf ace 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 a queous phase near the membrane interface however for the C-terminal ha lf of the peptide, residues were further off the membrane and were 10- 15 Angstrom 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 co nformations; however, side chains for these peptides penetrated the me mbrane-solution interface. We speculate that the relatively polar char acter of the N-terminal segment of Src and a Born repulsion energy pre vent this peptide from penetrating into the membrane interface when me mbrane bound.