title: Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.
abstract: During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle/Saale, Germany.|Biomolecular Mass Spectrometry and Proteomics, Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Philip
authors.initials: P
authors.lastname: Lössl
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle/Saale, Germany. andrea.sinz@pharmazie.uni-halle.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000588:Amines / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D019295:Computational Biology; D003432:Cross-Linking Reagents / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D046912:Multiprotein Complexes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods; D012984:Software
chemical_list: D000588:Amines; D000596:Amino Acids; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D011506:Proteins; D020543:Proteome
keywords: Chemical cross-linking; Mass spectrometry; Photo-affinity labeling; Photo-cross-linking; Protein 3D-structure; Protein interactions; Unnatural amino acids