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J Am Soc Mass Spectrom. 2013 Dec;24(12):1969-79. doi: 10.1007/s13361-013-0734-6. Epub 2013 Sep 12.

Structural analysis of guanylyl cyclase-activating protein-2 (GCAP-2) homodimer by stable isotope-labeling, chemical cross-linking, and mass spectrometry.

Author information

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

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.

PMID:
24026978
DOI:
10.1007/s13361-013-0734-6
[Indexed for MEDLINE]

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