title: Analysis of amyloid nanostructures using photo-cross-linking: in situ comparison of three widely used photo-cross-linkers.
abstract: Photoinduced cross-linking (PIC) has become a powerful tool in chemical biology for the identification and mapping of stable or transient interactions between biomacromolecules and their (unknown) ligands. However, the value of PIC for in vitro and in vivo structural proteomics can be realized only if cross-linking reports accurately on biomacromolecule secondary, tertiary, and quaternary structures with residue-specific resolution. Progress in this area requires rigorous and comparative studies of PIC reagents, but despite widespread use of PIC, these have rarely been performed. The use of PIC to report reliably on noncovalent structure is therefore limited, and its potentials have yet to be fully realized. In the present study, we compared the abilities of three probes, phenyl trifluoromethyldiazirine (TFMD), benzophenone (BP), and phenylazide (PA), to record structural information within a biomolecular complex. For this purpose, we employed a self-assembled amyloid-like peptide nanostructure as a tightly and specifically packed model environment in which to photolyze the reagents. Information about PIC products was gathered using mass spectrometry and ion mobility spectrometry, and the data were interpreted using a mechanism-oriented approach. While all three PIC groups appeared to generate information within the packed peptide environment, the data highlight technical limitations of BP and PA. On the other hand, TFMD displayed accuracy and generated straightforward results. Thus TFMD, with its robust and rapid photochemistry, was shown to be an ideal probe for cross-linking of peptide nanostructures. The implications of our findings for detailed analyses of complex systems, including those that are transiently populated, are discussed.
authors.affiliation: School of Chemistry, ‡Astbury Centre for Structural Molecular Biology, and §School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds , Woodhouse Lane, Leeds LS2 9JT, United Kingdom.
authors.forename: George W
authors.initials: GW
authors.lastname: Preston
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Wilson
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000648:ultrastructure*; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000648:ultrastructure*; D001386:Azides / Q000737:chemistry*; D001389:Azirines / Q000737:chemistry*; D001577:Benzophenones / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D010782:Photolysis; D014466:Ultraviolet Rays
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D001386:Azides; D001389:Azirines; D001577:Benzophenones; D003432:Cross-Linking Reagents; C470119:phenyl-3-trifluoromethyldiazirine; C014747:phenylazide; C047723:benzophenone