Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy

MARCKS-related protein (MRP) is a peripheral membrane protein whose binding to membranes is mediated by the N-terminal myristoyl moiety and a central, highly basic effector domain. MRP mediates cross-talk between protein kina se C and calmodulin and is thought to link the actin cytoskeleton to the pl asma membrane. Since MRP contains no tryptophan residues, we mutated a phen ylalanine in the effector domain to tryptophan (MRP F93W) and used fluoresc ence spectroscopy to monitor binding of the protein to phospholipid vesicle s. We report in detail the evaluation procedure necessary to extract quanti tative information from the raw data. The spectra of MRP F93W obtained in t he presence of increasing amounts of lipid crossed at an isosbestic point, indicating a simple transition between two states: free and membrane-bound protein. The change in fluorescence toward values typical of a more hydroph obic environment was used to quantify membrane binding. The partition coeff icient agreed well with values obtained previously by other methods. To stu dy the interaction of the N-terminus of MRP with membranes, a tryptophan re sidue was also introduced at position 4 (MRP S4W). Our data suggest that on ly the myristoylated N-terminus interacted with liposomes, These results de monstrate the versatility of site-directed incorporation of tryptophan resi dues to study protein-membrane interactions. (C) 2000 Academic Press.