title: Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by 2H solid-state NMR spectroscopy.
abstract: Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca2+ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca2+-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and 2H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the 2H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of -0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.
authors.affiliation: Junior Research Group Structural Biology of Membrane Proteins, Institute of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Vogel
authors.forename: Thomas
authors.initials: T
authors.lastname: Schröder
authors.forename: Christian
authors.initials: C
authors.lastname: Lange
authors.forename: Daniel
authors.initials: D
authors.lastname: Huster
mesh_terms: D003903:Deuterium; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D050356:Lipid Metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism
chemical_list: D051604:Guanylate Cyclase-Activating Proteins; D008565:Membrane Proteins; D019814:Myristic Acid; D003903:Deuterium