Caracterización morfológica y molecular de la proteína MARCKS en interfaces limpias y lipídicas

Tucuman

Congreso; XLI Reunión anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofisica

The Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein is enriched in membrane condensed domains (rafts), where it sequesters phosphatidyl inositol diphosphate (PIP2), mediating signaling involved in neural development. Lateral segregation of MARCKS/PIP2 is driven by non specific electrostatic/hydrophobic interactions of a highly basic Effector Domain in the protein (1). We study the molecular and interfacial properties of MARCKS as a context for other intrinsically unstructured proteins best known as oncoproteins. Unlike most biophysical studies of MARCKS, that use only the ED, we study the full length recombinant protein. We employed various techniques such as monolayers to assess viscoelastic properties and interaction with phospholipids (DPPS and DPPC), circular dicroism to determine conformation, and dynamic light scattering to evaluate aggregation. MARCKS shows low cut-off values with both lipids, although it establishes moderately attractive interactions with DPPC. Platforms for direct interaction with biological molecules were prepared by electrodeposited silver nanoparticles on crystalline silicon Si (111) substrates. Surface Enhancement of Raman Scattering (SERS) allows the molecular characterization and identification of adsorbed molecules on the platforms (2). Different programs of potential vs. time were applied in order to obtain a highly homogeneous and monodisperse distribution of SERS-active particles. Morphological characterization of these optimized platforms was performed with Scanning Electron Microscopy and their SERS activity was assessed through the vibrational spectra of Rhodamine B molecules. SERS spectra of DPPC monolayers transferred by Langmuir-Blodgett and Langmuir-Schaeffer methods, allows establishing the hydrophobic and hydrophilic interactions with the platform. Comparison of SERS spectra of disorderly deposited DPPC from aqueous solution give information of some features of the ordered monolayer. This methodology was also used to study mixed MARCKS/DPPC monolayers. 1- Denisov G, Wanaski S, Luan P, Glaser M, McLaughlin S. (1998). Biophys J. 74(2 Pt 1) 731. 2- Ivleva NP, Wagner M, Szkola A, Horn H, Niessner R, Haisch Ch. (2010) J. Phys. Chem. B 10184-10194. Agradecimientos: SECyT-UNC, ANPCyT, FONCyT and CONICET. GB and GL are CONICET researchers.