Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural i nformation on this protein, we have engineered 10 tryptophan residues betwe en positions 89 and 104 in the effector domain, a 24-residue-long amphipath ic: segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector doma in participates in the binding of MRP to CaM, as judged by the dramatic cha nges observed in the fluorescent properties of the mutants on complex forma tion. Intermolecular quenching of the fluorescence emission of the tryptoph an residues in MRP by selenomethionine residues engineered in CaM reveals t hat the N-terminal side of the effector domain contacts the C-terminal doma in of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properti es of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the i nteraction of MRP with CaM.