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.

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture

“…42 Thus, continued advances in MS methods coupled with fine-tuning cross-linker reactivities will undoubtedly enhance the utility of tandem covalent chemical capture-MS approaches. 43–46 Additionally, this study captured binding partners of Gal4 that rely upon the sequence surrounding position 849 for interaction. As there is considerable evidence that transcriptional activators use distinct but overlapping sequences within their activation domains to contact an array of binding partners, future experiments with a cross-linking amino acid placed at distinct positions will likely lead to the identification of additional coactivator targets.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…diazirine, benzophenone) (Fig. 7 d,e) can also be incorporated into crosslinker designs, which have an (essentially) non-specific side-chain reactivity (but do have some preferences) [176]. Cell permeable reagents can also be used to define the interfaces of protein assemblies in a cellular context [165,166].…”

Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers

“…MTS‐diazirine and MTS‐trifluoromethyl phenyl diazirine (MTS‐TFMD) (Figure a, Figure S1) were chosen as photoactivatable groups due to the superior performance of diazirines in comparative PI‐XL studies, their small size, and rapid, indiscriminate reactivity (Figure S2) . Both diazirine‐ and TFMD‐containing crosslinkers were synthesised as the photochemistry of TFMD leads to higher crosslinking efficiency, but it is bulkier .…”