MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic 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 domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties 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 interaction of MRP with CaM. MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity.

To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic 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 domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties 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 interaction of MRP with CaM. The proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family are protein kinase C substrates that have been proposed to regulate the actin cytoskeleton ().

The family comprises two members: MARCKS itself is a ubiquitous 32-kDa protein, whereas MARCKS-related protein (MRP, also called MacMARCKS) is a 22-kDa protein expressed mainly in brain and reproductive tissues (). Best songs pk download. MARCKS proteins share two conserved segments, namely the myristoylated N terminus and a central highly basic 24- to 25-residue-long segment, the effector domain, also called the “phosphorylation site domain” (, ). In vitro, MARCKS and MRP bind to calmodulin (CaM) with high affinity ( K d ≈ 5 nM) (, ). Although direct proof for an interaction between MARCKS proteins and CaM has so far not been obtained in cells, indirect evidence suggests that MARCKS proteins mediate crosstalk between the protein kinase C- and CaM-signal transduction pathways (for reviews, see refs. Two segments in MARCKS proteins are of interest with respect to their interactions with CaM. ( i) The Effector Domain.

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