Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.
MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.
['Tinoco|Luzineide W|LW|', 'Fraga|Jully L|JL|', 'Anobom|Cristiane D|CD|', 'Zolessi|Flavio R|FR|', 'Obal|Gonzalo|G|', 'Toledo|Andrea|A|', 'Pritsch|Otto|O|', 'Arruti|Cristina|C|']
D000595:Amino Acid Sequence D000818:Animals D000911:Antibodies, Monoclonal D001923:Brain Chemistry D002642:Chick Embryo D002942:Circular Dichroism D047908:Intracellular Signaling Peptides and Proteins D008565:Membrane Proteins D051379:Mice D008969:Molecular Sequence Data D000076250:Myristoylated Alanine-Rich C Kinase Substrate D010455:Peptides D010750:Phosphoproteins D010766:Phosphorylation D054730:Protein Interaction Domains and Motifs D011493:Protein Kinase C D017433:Protein Structure, Secondary D020349:Surface Plasmon Resonance
MARCKS Circular Dichroism Nuclear Magnetic Resonance Surface Plasmon Resonance Antibody binding Serine phosphorylation