A. Ulrich et al., Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy, P NAS US, 97(10), 2000, pp. 5191-5196
Citations number
40
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.