Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry |
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Authors: | Jens Pettelkau Iris Thondorf Stephan Theisgen Hauke Lilie Thomas Schröder Christian Arlt Christian H Ihling Andrea Sinz |
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Institution: | 1. Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120, Halle (Saale), Germany 2. Department of Technical Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, D-06120, Halle (Saale), Germany 3. Institute of Medical Physics and Biophysics, University of Leipzig, D-04107, Leipzig, Germany 4. Sandoz GmbH, Sandoz Biopharmaceuticals, Biochemiestra?e 10, A-6336, Langkampfen, Austria
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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 Ca2+. 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 Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-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. Figure ? |
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