title: Mapping protein structural changes by quantitative cross-linking.
abstract: Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.
authors.affiliation: Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Kukacka
authors.affiliation: Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Rosulek
authors.affiliation: Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Strohalm
authors.affiliation: Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.affiliation: Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic. Electronic address: pnovak@biomed.cas.cz.
authors.forename: Petr
authors.initials: P
authors.lastname: Novak
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000032:analysis* / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002147:Calmodulin; D003432:Cross-Linking Reagents
keywords: Chemical cross-linking; Mass spectrometry; Protein structure design; Proteolysis; Quantification