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Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

  • Research Article
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Journal of The American Society for 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 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.

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Acknowledgments

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, project Si 867/13-1) and the region of Sachsen-Anhalt.

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Author notes

  1. Thomas Schröder

    Present address: Sandoz GmbH, Sandoz Biopharmaceuticals, Biochemiestraße 10, A-6336, Langkampfen, Austria

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120, Halle (Saale), Germany

    Jens Pettelkau, Christian Arlt, Christian H. Ihling & Andrea Sinz

  2. Department of Technical Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, D-06120, Halle (Saale), Germany

    Iris Thondorf, Hauke Lilie & Thomas Schröder

  3. Institute of Medical Physics and Biophysics, University of Leipzig, D-04107, Leipzig, Germany

    Stephan Theisgen

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Correspondence to Andrea Sinz.

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Pettelkau, J., Thondorf, I., Theisgen, S. et al. Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry. J. Am. Soc. Mass Spectrom. 24, 1969–1979 (2013). https://doi.org/10.1007/s13361-013-0734-6

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