Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Wednesday Speaker Abstracts

Biophysical Studies of Myristoylated Unique and SH3 Domains of Src Kinase and their Interaction with Lipid Membranes Anabel-Lise Le Roux 2,1 , Borja Mateos López 1 , Bruno Castro 3 , Maria-Antonia Busquets Viñas 1 , Maria Gracia Parajo 3 , Francesc Sagués 1 , Miquel Pons 1 . 1 University of Barcelona, Barcelona, Spain, 2 Institute for Research in Biomedicine, Barcelona, Spain, 3 Institute of Photonic Sciences, Castelldefels, Spain. C-Src is a member of the Src family of non-receptor tyrosine kinases, which are involved in many signaling pathways. It is composed of the N-terminal, the SH3, SH2, kinase and C- terminal domains, and is anchored to membranes via cooperative electrostatic and hydrophobic interactions. Indeed the N-terminal intrinsically disordered region (Unique Domain UD) is myristoylated. Weak interactions with lipids in the Unique and SH3 domains and intramolecular interactions between them were recently found in the non-myristoylated form. Our objective consisted in characterizing the myristoylated form of the Unique + SH3 domain (MyrUSH3). Binding kinetics to liposomes was followed using surface plasmon resonance (SPR) and revealed two MyrUSH3 populations, a dominant form binding with fast association and dissociation, and a minor persistently bound (PB) population not described earlier, found to possibly be MyrUSH3 dimers. In a construct in which the SH3 domain was replaced by the GFP protein, single molecule photobleaching experiments of these PB species bound to liposomes were conducted. A major population of dimers over the bilayer surface was detected. Lower affinity lipid binding regions in the UD and SH3 domains were studied by nuclear magnetic resonance (NMR), as well as their intermolecular interactions. The latter were conserved in presence of the myristoyl chain, but secondary lipid binding regions were found to behave differently in presence or absence of the acyl moeity. The SPR and fluorescence studies revealed autoassociation tendencies of the myristoylated N-terminal domain of c-Src upon binding to liposomes. NMR data highlighted the interplay between the lipid biding regions of UD and SH3 and the intermolecular interactions and revealed a myristoyl interacting loop in SH3. Thus, the myristoylated intrinsically disordered UD may act in c-Src regulation at the lipid bilayer interface.

QCM-D: A Powerful Surface Analysis Tool for Protein Studies Jennie Ringberg Biolin, Sweden No Abstract

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