Photo-induced covalent cross-linking for the analysis of biomolecular interactions

Preston, George W.; Wilson, Andrew J.

doi:10.1039/c3cs35459h

Cited by 177 publications

(200 citation statements)

References 57 publications

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“…BP can be manipulated in ambient light and activated at λ ≈360 nm. It is chemically stable and reacts preferentially with unreactive C−H bonds, which is widely used to study protein–protein as well as protein–RNA interactions . The substituents on BP can affect the photochemistry significantly and electron‐withdrawing groups increase the efficiency of H‐abstraction .…”

Section: Methodsmentioning

confidence: 99%

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

et al. 2019

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Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring‐nitrogen methylation, such as m7G and m1A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α‐hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho‐nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site‐specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m7G and m1A with spatiotemporal control.

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Section: Methodsmentioning

confidence: 99%

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

et al. 2019

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“…PA generates a highly reactive nitrene that rearranges to an intermediate with strong reactivity preferences. 13,18 Given their commercial availability, PA and BP have been employed widely in PIC studies of protein interactions, despite misgivings about their suitability for extracting molecular-level information. For BP the caveats include the potential for cross-linking chemistry to be determined by diffusion rather than supramolecular structure, while for PA cross-links can be templated by covalent preference rather than noncovalent structure.…”

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confidence: 99%

Analysis of Amyloid Nanostructures Using Photo-cross-linking: In Situ Comparison of Three Widely Used Photo-cross-linkers

et al. 2014

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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.

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“…It has been particularly useful in the analysis of molecular interactions in the transition state or weak binding state, which otherwise would be difficult to access using the X-ray crystallography and NMR techniques. 4 In general, identification of the labeled sites has been achieved using tandem MS-based sequencing of purified and labeled peptides after digestion. To get better performance of PAL-based target identification, many efforts have been made aiming to purify negligible amounts of the labeled products from an enormous concomitant using several separation tags such as a biotin, 5 a perfluoroalkyl group, 6 a clickable tag.…”

Section: Introductionmentioning

confidence: 99%