title: The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺.
abstract: Guanylate cyclase activating protein-2 (GCAP-2) is a Ca²⁺-binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺-free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca²⁺-dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca²⁺-free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺-myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca²⁺-independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca²⁺-bound and Ca²⁺-free forms, respectively, to gain information on the nature of the Ca²⁺-dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca²⁺-free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺-bound state.
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle 06120, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schröder
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Christian
authors.initials: C
authors.lastname: Lange
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019814:Myristic Acid / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D013696:Temperature; D013816:Thermodynamics
chemical_list: D051604:Guanylate Cyclase-Activating Proteins; D019814:Myristic Acid; D002118:Calcium