title: Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles.
abstract: GCAPs are neuronal Ca(2+)-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but (2)H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca(2+)-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca(2+) and membrane mimetics.
authors: Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia; Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors: Aleksandra
authors: A
authors: Margetić
authors: Center for Structural Biology, Vanderbilt University, 465 21st Ave South, Nashville, TN, USA.
authors: David
authors: D
authors: Nannemann
authors: Center for Structural Biology, Vanderbilt University, 465 21st Ave South, Nashville, TN, USA.
authors: Jens
authors: J
authors: Meiler
authors: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany; Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India. Electronic address: daniel.huster@medizin.uni-leipzig.de.
authors: Daniel
authors: D
authors: Huster
authors: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors: Stephan
authors: S
authors: Theisgen
keywords: GCAP-2; ROSETTA; bicelles; membrane-protein interaction; myristoyl switch; solution NMR