title: Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.
abstract: 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.
authors.affiliation: Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS, Bloco H, Rio de Janeiro 21941-902, RJ, Brazil. Electronic address: lwtinoco@nppn.ufrj.br.
authors.forename: Luzineide W
authors.initials: LW
authors.lastname: Tinoco
authors.affiliation: Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS, Bloco H, Rio de Janeiro 21941-902, RJ, Brazil. Electronic address: jully.lfraga@gmail.com.
authors.forename: Jully L
authors.initials: JL
authors.lastname: Fraga
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, CT, Bloco A, Rio de Janeiro 21941-909, RJ, Brazil. Electronic address: anobom@iq.ufrj.br.
authors.forename: Cristiane D
authors.initials: CD
authors.lastname: Anobom
authors.affiliation: Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. Electronic address: fzolessi@fcien.edu.uy.
authors.forename: Flavio R
authors.initials: FR
authors.lastname: Zolessi
authors.affiliation: Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay. Electronic address: gobal@pasteur.edu.uy.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Obal
authors.affiliation: Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. Electronic address: atoledo@fcien.edu.uy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Toledo
authors.affiliation: Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay. Electronic address: pritsch@pasteur.edu.uy.
authors.forename: Otto
authors.initials: O
authors.lastname: Pritsch
authors.affiliation: Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. Electronic address: arruti@fcien.edu.uy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Arruti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001923:Brain Chemistry; D002642:Chick Embryo; D002942:Circular Dichroism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C
keywords: Antibody binding; Circular Dichroism; MARCKS; Nuclear Magnetic Resonance; Serine phosphorylation; Surface Plasmon Resonance