title: Structural analysis of guanylyl cyclase-activating protein-2 (GCAP-2) homodimer by stable isotope-labeling, chemical cross-linking, and mass spectrometry.
abstract: The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca(2+). In-depth MS and MS/MS analysis of the cross-linked products was aided by (15)N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca(2+)-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca(2+)-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120, Halle (Saale), Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Pettelkau
authors.forename: Iris
authors.initials: I
authors.lastname: Thondorf
authors.forename: Stephan
authors.initials: S
authors.lastname: Theisgen
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Thomas
authors.initials: T
authors.lastname: Schröder
authors.forename: Christian
authors.initials: C
authors.lastname: Arlt
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D062105:Molecular Docking Simulation; D009587:Nitrogen Isotopes / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D053719:Tandem Mass Spectrometry
chemical_list: D003432:Cross-Linking Reagents; D051604:Guanylate Cyclase-Activating Proteins; D009587:Nitrogen Isotopes; D002118:Calcium
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